EFFECTS OF 
ORGANIC 
ACIDS,
 
HOP ACID
S
 
AND 
THEIR 
MIXTURES ON 
 
THE 
INHIBITION
 
OF
 
LISTERIA MONOCYTOGENES
 
By
 
Thanikarn Sansawat
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
A DISSERTATION
 
Submitted to
 
Michigan State University
 
in partial fulfillment of the requirements
 
f
or the 
degree of
 
Food Science 
-
 
Doctor of Philosophy
 
2015
ABSTRACT
 
EFFECTS OF ORGANIC 
ACIDS,
 
HOP ACID
S
 
AND 
THEIR 
MIXTURES ON 
 
THE 
INHIBITION
 
OF
 
LISTERIA MONOCYTOGENES
 
By
 
Thanikarn Sansawat
 
 
L
isteria
 
monocytogenes 
is responsible for an infectious disease called l
isteriosis
,
 
which 
occurred
 
after
 
the consumption of 
foods
.
  
Among 
various
 
foods, meat products such as 
RTE 
meats, fr
ankfurters, deli meat, and pate
 
have been ranked first as the types of food vehicles 
involved in listeriosis outbreaks due to the extended s
torage and frequent consumption with no 
additional heat
.  
T
he overall goal of t
his study 
was to 
investigate the 
antilisterial 
effect
s
 
of 
different organic acid salts, hop acid extracts, and their comb
inations in liquid media and 
processed meats.  To achiev
e the overall goal, four separated studies were conducted.    
 
In study I, 
nine different organic acid salts were investigated for 
Listeria
 
inhibition,
 
physicochemical changes and 
organolepti
c characteristics 
in
 
full
-
 
and low
-
sodium frankfurters
.  
P
otassi
um acetate and potassium diacetate (P
APD
)
 
out 
of 
the 
nine organic acid mixtures was the 
most
 
effective in inhibiting 
Listeria 
in 
full
-
 
and low
-
sodium 
frankfurters during storage at 4, 7 or 
10
o
C.  The sensory characteristics of all formulations were similar
 
except a low score
 
was 
seen 
for flavor and overall acceptability in low
-
sodium frankfurters containing PAPD. 
 
In study II, 
eight different hop acid extracts 
were investigated for 
Listeria
 
inhibition 
with/without 
PAPD
 
in trypticase soy broth containing yea
st extract (TSBYE)
.  
Five hop acid
 
extract
s
 

-

-
acid, acid
-
tetra, K
-
tetra, and K
-
hexa) 
out of 
the 
eight including acid
-
iso, K
-
iso, and K
-
rho 
significantly inhibited 
Listeria
 
in liquid media 
at 25 and 50 ppm 
at
 
37
o
C
.  The 
combinations o
f these five hop acids at 25 or
 
50 ppm with 0.5% PAPD led
 
to better inhibition of
 
Listeria
 
than any
 
single hop acid or 0.5% PAPD
 
alone
.  After 30 min exposure at 
85
o
C, all 
of the 
five 
hop acids were heat stable with the best inhibitory activity seen for 
the 

-
acid, regardless of 
heating time.  
At 7
o
C in liquid m
edia, 
the mixture of 
5 ppm hop acid/0.5% PAPD was
 
listeristatic, whereas none of the single hop acids showed any 
Listeria 
inhibition 

-
acid.  
 
 
In study III, 
the
 
antilisterial activity of hop 

-
 

-
acids 
at 5 ppm 
with/without 0.5% 
PAPD 
were inves
tigated 
in deli
-
style turkey meats during storage 
at 4 and 7
o
C
.
  
B
oth 

-
 

-
acids
 
at 5 ppm 
did not inhibit 
L. monocytogenes 
during storage at 4 and 7
o
C,
 
while the 
hop/PAPD combinations were listeristatic, regardless of the hop acid type.  Similar result
s of no 
inhibition were observed in skim milk and 2% milk containing 

-
 
or
 

-
acid
 
at 5 ppm.  
 
 
 
In study IV, 
antilisterial activities of 

-
 

-
acids 
at various concentrations were 
investigated 
in turkey slurry at 7 and 37
o
C.
  
Hop 

-
 

-
acids exhi
bited antilisterial activity at 
the 
concentration 
>
 
750 ppm 
at 3
7
o
C 
and 
>
 
500 ppm at 
7
o
C
, respectively.  
These results indicate 
that formulation of single hop acid 
required > 500 ppm at 
7
o
C to inhibit 
Listeria
 
in meat 
products, which is 100 or more times 
g
reater 
than 
the 5 ppm hop acid 
in
 
liquid media
.
 
 
  
 
iv
 
 
ACNOWLEDGEMENTS
 
 
I would like to acknowledge all 
of the 
people who
 
contributed to this dissertation.  
Even
 
a
 
million words are not enough to 
express how much I appreciate
 
their 
contributions to 
my 
success
.
  
First
 
and foremost
, I want to
 
express
 
my sinc
ere gratitude to Dr. Ike Kang.  
I thank him
 
for 
giving me the oppo
rtunity to conduct this research
.  
Dr. Ka
ng was not only my academic 
advisor
, but 
the
 
person treating
 
me like his daughter
.  
Dr. Kan
g 
helped me in every way he could,
 
and always encouraged me to think positively
,
 
even at
 
the darkest 
and toughest 
moment
.  
Without Dr. Kang,
 
I knew that 
this dissertation would 
not be 
possible.  He is
 
the person I will 
respect 
for 
the entire of my 
life.
  
I
 
sincere
ly 
thank my committee members Dr. Gale Strasburg, 
Dr. El
l
iot Ryser, Dr. Bradl
e
y Marks, and Dr. Darrin Karcher
 
for their 
valuable 
time, 
useful 
assist
ance
, 
helpful discussions, 
and 
full
 
support
.
  
With
out their guidance, I will not be
 
able to get 
thro
ugh the challenging
 
obstacles
,
 
and complete this project
.
 
 
I also
 
thank my lab colleag
u
es
, 
technicians,
 
and 
the 
MSU 
meat lab team
 
for their
 
willingness to
 
hel
p 
make this project possible.  
In addition to those mentioned above, I 
thank my frien
ds who 
shared
 
the 
good and bad times, 
and 
mad
e me feel warm 
like I was
 
at
 
home
,
 
although I was
 
staying 
far away from my home.  
Most
 
i
mportantly, I 
deeply tha
nk my family who always ga
ve 
me plenty of unconditional love, 
and never let me give up
, e
specially my mom
 
who wa
s always there for me
.
 
 
 
 
 
 
v
 
 
TABLE OF CONTENTS
 
 

 

 
x
 


 
1
 

 
5
 
1.1
 
Lis
teria monocytogenes

 
5
 
1.1.1
 
Characteristics of 
Listeria monocytogenes

 
5
 
1.1.2
 

 
6
 
1.1.3
 
Listeriosis outbreaks and incidences of 
Listeria monocytogenes 
in RTE
 


 
6
 
1.1.4
 
USDA guidelines to control 
Listeria monocytogenes
 
in RTE meat
 

 
9
 
1.2
 

 
11
 
1.2.1
 

 
11
 
1.2.2
 


 
13
 
1.2.3
 

 
14
 
1.3
 

 
17
 
1.3.1
 

 
17
 
1.3.2
 

 
18
 
1.3.3
 

 
21
 
CHA
PTER 
2:
 
INHIBITION OF 
LISTERIA MONOCYTOGENES
 
IN FULL AND LOW 
SODIUM FRANKFURTERS AT 4, 7, OR 10ºC USING SPRAY
-
DRIED 
MIXTURES OF ORGANIC ACIDS SALTS

 
24
 
2.1
 

 
24
 
2.2
 

 
24
 
2.2.1
 
Full sodium frankfurter preparation using powdered or liquid 

 
25
 
2.2.2
 
Low sodium frankfurter preparation with powdered or liquid
 

 
26
 
2.2.3
 

 
30
 
2.2.4
 
List
eria 
monocytogenes
 

 
30
 
2.2.5
 

 
31
 
2.2.6
 
Consumer sensory analysis, full
-

 
32
 
2.2.7
 
Consumer sensory analysis, low
-

 
33
 
2.2.8
 
St

 
33
 
2.3
 

 
34
 
2.3.1
 

 
34
 
2.3.2
 

 
36
 
2.3.3
 

 
44
 
vi
 
 
2.4
 

 
47
 
CHAPTER 
3:
 
ANTILISTERIAL EFFECTS OF DIFFERENT HOP ACIDS IN 
COMBINATION WITH POTASSIUM ACETATE AND POTASSIUM 
DIACETATE AT 7 AND 37ºC

 
48
 
3.1
 

 
48
 
3.2
 
Materia

 
48
 
3.2.1
 
H

 
48
 
3.2.2
 
L. monocytogenes 

 
50
 
3.2.3
 
Antilisterial activity of eight hop extracts at 37
o

 
50
 
3.2.4
 
De
termination of synergistic effect of hop acid/PAPD mixtures on 
inhibition of 
L. monocytogenes 
at 37
o

 
50
 
3.2.5
 

 
51
 
3.2.6
 
Minimal inhibitory concentrations (MIC) of hop extractions in TSBYE.
 
51
 
3.2.7
 
Antilisterial activity of hop extracts and PAPD mixtures at 7
o

 
51
 
3.2.8
 

 
52
 
3.3
 

 
53
 
3.3.1
 
Inhibitory activity of hop acids against 
L. monocytogenes 
at 37
o

 
53
 
3.3.2
 
Sy
nergistic effect of hop acid/PAPD mixtures on inhibition of 
L.
 
monocytogenes 
at 37
o

 
54
 
3.3.3
 
Thermal stability of hop acid with/without PAPD at 85
o

 
56
 
3.3.4
 
Minimal inhibitory concentrations of hop acids against 
Listeria 
growth at
 
37
o
C

 
59
 
3.3.5
 
Effect of hop acid/PAPD mixtures on inhibition of 
L.
 
monocytogenes 
at 7
o

 
60
 
3.4
 

 
63
 
CHAPTER 
4:
 
INHIBITION OF 
LISTERIA MONOCYTOGENES
 
IN DELI
-
STYLE TURKEY 
AND MILK USI
NG HOP ACID EXTRACTS WITH OR WITHOUT 

 
64
 
4.1
 

 
64
 
4.2
 

 
65
 
4.2.1
 
Deli
-
style turkey preparation with/without 
Listeria
 


 
65
 
4.2.2
 
Physicochemical analysis of deli turkey
 
meat

 
66
 
4.2.3
 
Listeria monocytogenes
 

 
67
 
4.2.4
 

 
67
 
4.2.5
 

 
68
 
4.2.6
 
Antilisterial activit
y of hop extracts and PAPD mixtures in milk at 7
o
C..
 
68
 
4.2.7
 

 
69
 
4.3
 

 
69
 
4.3.1
 

 
69
 
4.3.2
 
L. monocytogenes
 


 
71
 
4.4
 

 
80
 
CHAPTER 
5:
  
ANTILISTERIAL EFFECT OF HOP ALPHA AND BETA ACIDS IN TURKEY 
SLURRY AT 7 AND 37ºC 

 
81
 
vii
 
 
5.1
 

 
81
 
5.2
 


 
81
 
5.2.1
 
Preparation of hop acids (alpha and beta) and 
L. monocytogenes 
strains..
 
81
 
5.2.2
 

 
82
 
5.2.3
 
Antilisterial activity of hop extracts at 7 and 37
o

 
82
 
5.2.4
 


 
83
 
5.3
 

 
83
 
5.3.1
 
Antilisterial activity of hop acids at 37
o

 
83
 
5.3.2
 
Antilisterial activity of hop acids at 7
o

 
85
 
5.4
 

 
89
 


 
90
 
FUTURE RECOMMENDATIONS...

 
91
 

 
92
 
 
 
APPENDI
X A: Tables of supplemental data


 
93
 
 
APPENDIX B: 
Calculatio



.


 
106
 

 
109
 
 
 
 
 
 
 
 
 
 
 
 
 
 
viii
 
 
LIST OF TABLES
 
 
Table 1.1
 
 

8
 
 
Table 1.2
 

 
19
 
 
Table 2.1  
 
Base formulation of
 
fran

 
27
 
 
Table 2.2  
 
Components of seasoning blend (no salt) added to
 

 
28
 
 
Table 2.3  
 
Percent of powdered inhibitors (PI) and liquid inhibitors (LI) added to frankfurter
 
 
 
formulations


 
29
 
 
Table 2.4  
 
Impact of formulations containing powdered and liquid inhibitors on the 
 
physicochemical properties of full 

 
35
 
 
Table 2.5  
 
Impact of formulations containing powdered and liquid inhibitors on t
he
 
 
 
physicochemical properties of low so

 
36
 
 
Table 2.6 
 
Impact
 
of powdered and liquid inhibitors on sensory properties of full sodium 
 


 
46
 
 
Table 2.7 
 
Impact of powdered and liquid inh
ibitors on consumer acceptance scores in low 
 
sodium frankfurt

 
46
 
 
 
Table 3.1
  
 
Concentrations of eight hop acid extracts and potassium acetate/potassium 
diacetate

 
49
 
 
 
 
Table 3.2
  
 
Interpretation e
ffects of 












 

 
 
Table 3.3  
 
Population of 
L. monocytogenes
 
in TSBYE containing 25 ppm hop acid extracts 
with/without 0.5% PAPD a
fter heating at 85
o

 
58
 
 
Table 3.4  
 
Minimal inhibitory concentrations (ppm) of hop acid extracts on 
L. 
monocytogenes 
grow

 
59
 
 
Table 4.1  
 
Physicochemical properties of deli
-
s

 
70
 
 
Ta
ble 5.1  
 
Population of 
L. monocytogenes 
(log CFU/g)
 
in turkey slurries containing 0 to 
1000 ppm 

-
acid
 

-
acid
 
after incubating at 37
o

 
84
 
Table A.1 
 
Population of 
L. monocytogenes
 
on vacuum
-
packaged full
-
sodium frankfurters 
with powdered or liquid inhibitors during 90 days
 

 
94
 
ix
 
 
Table A.2
 
Area under gr
aph 
of 
L. monocytogenes 
population 
on vacuum
-
packaged full 
sodium frankfurters with powdered or liquid inhibitors during 90 days of storage 


 
95
 
 
Table A.3 
 
Population of 
L. monocytogenes
 
on vacuum
-
packaged lo
w
-
sodium frankfurters 
with powdered or liquid inhibitors during 90 days of stor

 
96
 
Table A.4
 
Area under graph 
of 
L. monocytogenes 
population 
on vacuum
-
packaged low 
sodium frankfurters with powdered or liquid inhibitors during 90 days o
f storage 
at 4, 7 and 10ºC

 
96
 
 
Table A.5 
 
Population 
of mesophilic aerobic bacteria on vacuum
-
packaged frankfurters with 
powdered or liquid inhibitors during 90 days of sto

 
.97
 
Table A.6  
 
Population
 
of mesophilic aerobic bacteria on vacuum
-
packaged low
-
sodium 
frankfurters with powdered or liquid 
Listeria
 
growth inhibitors during 90 days of 
storage at 4, 7 and 

 
Table A.7
  
 
Population of 
L. monocytogenes 
in TSBYE with or 
without different hop acid 
extracts at 5 ppm
, 
0.5 or 1% PAPD, 


 
during 6 days of storage at 7
o

 
99
 
Table A.8  
 
Population of 
L. monocytogenes 
on vacuum
-
packaged deli
-
style turkey meat with 
various
 
inhibitors during 60 days of storage at 4 and 
7
o

 
100
 
Table A.9  
 
Population of 
L. monocytogenes 
at 4 and 
7
o
C 
on aerobic
-
packaged deli
-
style 
turkey meat with various inhibitors and sliced after 30 days of storage whole 
sticks



 
101
 
Table A.10  
 
Population of 
L. monocytogenes 
at 4 and 
7
o
C 
on aerobic
-
packaged deli
-
style 
turkey meat with various inhibitors and sliced after 60 days of storage whole 
sticks

.
 
102
 
Table A.11  
 
Population of 
L.
 
monocytogenes 
(log CFU/mL) in skim milk with or without 
different hop acid extracts at 5 ppm or 0.5% PAPD during 6 days of storage at 
7
o


 
103
 
Table A.12  
 
Population of 
L. monocytogenes 
(log CFU/mL) in 2% milk with or witho
ut 
different hop acid extracts at 5 ppm or 0.5% PAPD during 6 days of storage at 
7
o


 
104
 
Table A.13 
 
 
Population of 
L. monocytogenes 
(log CFU/g)
 
in 
turkey slurries
 
containing 

-
acid
 

-
acid
 
at 0 to 1000 ppm 
during 12 days 
of storage at 7
o

 
105
 
Table B.1  
 
Interpretation possible effects of 










 

 
x
 
 
LIST OF FIG
URES
 
 
Figure 1.1
 

 
12
 
 
Figure 1.2  
 

 
13
 
 
Figure 1.3  
 
Some hop compounds and reduced iso
-
alpha
-


 
20
 
 
Figure 1.4  
 

 
22
 
 
Figure 2.1
 
Population of 
L. monocytogenes
 
on vacuum
-
packaged full
-
sodium frankfurters 
formulated with powdered or liquid inhibitors during 90 days of storage at 4, 7 
and 10
o


 
40
 
 
Figure 2.2
 
Population of 
L. monocytogenes
 
on vacuum
-
packaged low
-
sodium frankfurters 
formulated with powdered or liquid inhibitors during 90 days of storage at 4, 7 
and 10
o


 
41
 
 
Figure 2.3
 
Population of mesophilic aerobic bacteria on vacuum
-
packaged full
-
sodium 
frankfurters formulated with powdered or liquid inhibitors
 
during 90 days of 
storage at 4, 7 and 10
o

 
42
 
 
Figure 2.4
 
Population of mesophilic aerobic bacter
ia on vacuum
-
packaged low
-
sodium 
frankfurters formulated with powdered or liquid inhibitors
a
 
during 90 days of 
storage at 4, 7 and 10
o

 
43
   
 
Figure 3.1

 











 

 
 
Figure 3.2
  
 















 

 
 


 
Population of 
L. monocytogenes  
in TSBYE with or without different hop acid 
extracts at 5 ppm
, 
0.5 or 1% PAPD, 


 
during 6 days of storage at 7
o

..
 
61
 
 
Figure 4.1  
 
Expected
 
times for production, distribution, and consumption of delicatessen 


 
65
 
 
Figure 4.2
 
L. monocytogenes 
populations
 
in vacuum
-
packaged deli
-
style turkey meat with 
various inhibitors during 60 da
ys of storage at 4 and 
7
o

 
73
 
 
xi
 
 
Figure 4.3
 
L. monocytogenes 
populations
 
during 10 days of storage at 4 and 
7
o
C 
in aerobic
-
packaged deli
-
style turkey meat with various inhibitors and sliced after 30 days of 


 
 
Figure 4.4
 
L. monocytogenes 
populations
 
during 10 days of storage at 4 and 
7
o
C 
in aerobic
-
packaged deli
-
style turkey meat with various inhibitors and sliced after 60 days of 


 
76
 
 
Figure 4.5
 
L. monocytogenes 
populatio
ns
 
in skim milk and 2% milk with or without different 
hop acid extracts at 5 ppm or 0.5% PAPD during 6 days of storage at 7
o

 
79
 
 
Figure 5.1
 
Population of 
L. monocytogenes 
in 
turkey slurries
 
containing 0 to 1000 ppm 

-

-
acid during 12 days of storage at 7
o

 
1
 
 
INTRODUCTION
 
 
Listeria monocytogen
e
s
 
is readily 
destroyed
 
during normal 
cooking
 
(Zaika et al., 1990)
, 
but 
the pathogen 
is
 
of greatest concern as a
 
post
-
thermal 
contaminant 
especially in 
meats due to 
its ability to
 
grow durin
g extended refrigerated storage (James et al., 1985; Sauders and 
Weidmann, 2007; Adam and Moss, 2008; Tompkin, 2002; USDA
-
FSIS, 1999a,b)
.
 
 
As a result,
 
the 
consumption of 
Listeria
-
contaminated foods 
such as ready
-
to
-
ea
t (RTE) meat 
could cause
 
ho
spitalizations with
 
high rates of death (Stacy et al., 2014
; Cartwright et al., 2013
).  
A
mong 23 
food categories
,
 
b
oth deli meats and frankfurters (non
-
reheated) are associ
ated with 
greater risk 
of
 
listeriosis (
FDA/CFSAN 
and USDA
/FSIS, 2003).
  
Consumption of contaminated turkey 
frankfurters was linked to a massive multi
-
state outbreak during 1998
-
1999 that involved 108 
cases, including 14 fatalities an
d 4 miscarriages or stillbirths (Mead et al., 2006)
. 
 
C
onsumption 
of non
-
reheate
d frankfurters ranked second and fourth in terms of risk on a per serving 
basis and 
an
 
annual basis, respectively
, according to 
the 2003 
Listeria
 
risk assessm
ent of ready
-
to
-
eat 
(RTE) foods (
USDA/FSIS, 2003
c
)
.
  
Based on the recent report from
 
the Centers f
or Disease 
Control and Prevention (CDC), the incidence of listeriosis in 2013 has not decreased, compared 
with 2010 

 
2012, indicating a gap between the current food saf
ety system and the need for 
better
 
food safety interventions (Crim et al., 2014).  
 
 
T
o
 
control and minimize both 
presence and levels of 
L. monocytogenes 
in RTE meat and 
poultry products
, 
U.S. Department of Agriculture, Food Safety and Inspection Service 
(USDA/FSIS)
 
issued an interim final rule requiring one of three 
alternatives: (i) apply 
both a 
post
-
lethality treatment and an antimicrobial agent; (ii) apply either a post
-
lethality treatment or 
antimicrobial agent; or (iii) use of sanitation control measures to prevent recontamination after 
2
 
 
processing (USDA/FSIS, 2003
a
).
 
 
Many
 
Listeria
 
cont
rol strategies have been assessed
,
 
including 
the addition of sodium lactate and/or diacetate to
 
the product formulation (Bedie et al., 2001; 
Glass et al, 2002; Hwang and Tamplin, 2007; Legan et al,2004; Lianou et al, 2007a
,
b; 
Luchansky et al, 2006; Mbandi 
and Shelef, 2002; Pradhan et al, 2009; Samelis, et al, 2005; 
Seman, et al, 2002; Stekelenburg, 2003; Stekelenburg, 2001; Uhart, et al, 2004)
 
application of 
steam 
or/and hot water pasteurization (Lecompte et al, 2008; Murphy et al, 2003; Murphy et 
al,2002; 
Murphy et al, 2006;Murphy et al, 2005; Sommers et al, 2002), irradiation (Chun et al, 
2009; Foong et al, 2004; Gursel and Gurakan, 1997; Jin et al, 2009; Zhu et al, 2009)
, and high 
pressure 
processing (Basaran
-
Akgul et al, 2010; Bowman et al, 2008; Gudbjom
sdottir et al, 
2010)
. 
 
Some of these approaches 
have marginal success
 
because
 
of 
organoleptic concerns 
about
 
sodium lac
tate
-
diacetate (Blom et al, 1997; Islam et al, 2002)
, quality, cost and consumer 
acceptability issues
 
about
 
food 
irradiation (Foong et al
, 2004)
, high capital investment and 
low 
throughput for high
-
pressure processing
 
(Balasubramaniam and Farkas, 2008)
,
 
and fat smearing 
and/or purging for steam and 
hot water pasteurization 
(personal observation
).
  
Hence, 
additional 
research is still needed 
to develop new 
inhibitors or improve 
current food additives for the 
inhibition
 
of 
L. monocytogenes.
 
 
  
Currently, a
ntilisterial agents are 
commonly used by manufacturers of RTE meat 
products with a wide range of choices (Aureli et al., 1992; Tassou et al.,
 
1995; Thongson et al., 
2005; Lucus and Were, 2009).  
According to the Interagency Risk Assessment for 
L. 
monocytogenes 
in retail delicatessens, the predicted risk of listeriosis from the consumption of 
RTE deli products could be reduced by approximately 9
6% if those products contain 
antimicrobial agents (Akingbade et al., 2013).
  
The application of various organic acids and their 
salts 
to RTE meats is effective on
 
inhibiting 
Listeria 
growth (Barmpalia et al., 2004; Bedie et al., 
3
 
 
2001; Blom et al., 1997; Gl
ass et al., 2002; Hwang et al., 2007; Islam et al., 2002; Mbandi and 
Shelef, 2002; Samelis et al., 2005; Seman et al., 2008; Stekelenburg, 2003).  However, most 
organic acids negatively affect both flavor and organoleptic 
taste
 
(
Blom et al, 1997; Islam et 
al, 
2002)
.  Hence, 
the reduction of negative impacts from organic acids 
w
hile maintaining or 
improving the antilisterial activity 
have been evaluated based on the combined effect of 
Listeria 
inhibition 
(Barmpalia et al., 2004; Glass et al., 2002; Mbandi an
d Shelef, 2001; Stekelenburg, 
2003).  
 
 
  
 
Hop acids, used in beer industry, have 
been known to possess 
antimicrobial activities 
against gram
-
positive bacteria (Haas and Barsoumian, 1994; Bhattacharya et al., 2003
; Sakamoto 
and Konings, 2003)
.  

-
 

-
 
(lupulone)
 
acids
 
are major hop resin 
compo
nents, which give damage to 
bacterial cytoplasmic membrane, interfere with active 
transport of sugar 
and amino acids, and reduce 
intracellular pH by dissociating into protons and 
hop a
nions (Teuber and Schmalreck, 1973;
 
Simpson and Hammond 1991; Blanco et al. 2006). 
 
Hop extracts are more popular than the traditionally dried hops due to the convenience, stability, 
economic cost, and good 
quality (Wilson et al., 2003).  
U
ntil
 
today, howe
ver, only a 
few studies 
have been 
conducted 
to evaluate the effects of
 
hop/organic acid mixtures
 
on 
Listeria 
inhibition
 
i
n 
meat products.
 
 
Therefore, 
the overall goal of 
this study 
wa
s 
to 
evaluate
 
the 
antilisterial activity
 
of 
different 
organic acids
 
and 
h
op acids, and 
the
 
mixture of the
 
best organic acid and 
hop acid
 
components
.  
 
In this study, we hypothesized
 
that 
combination
s
 
of hop acid/organic acid
 
effectively 
inhibit
 
the growth of 
L. monocytogenes 
in RTE meat products.  
To achieve this
 
goal, 
the spec
ific 
objectives of this study were
 
to
: 
(1) 
assess 
L.
 
monocytogenes
 
inhibition 
and the physicochemical 
and 
organolepti
c characteristics 
of full
-
 
and low
-
sodium frankfurters
 
prepared with nine different 
4
 
 
organic acid salts
,
 
(2) 
assess 
the 
antilisterial effect
 
of eight
 
hop 
acid 
extracts
 
with
/without
 
the 
best organic acid salts 
in 
TSBYE, (3)
 
assess 
the 
antilisterial activity of the best hop 
acid 
and 
the 
best organic acid
 
in deli
-
style turkey meats 
during storage
 
at 4 and 
7
o
C
, and (4) 
assess 
the 
antilisterial act
ivities of 
the best hop and organic acids
 
in turkey slurry at 7 and 37
o
C.
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5
 
 
CHAPTER 1
: 
LITERATURE REVIEW
 
 
1.1
 
 
Listeria monocytogenes
  
 
Listeria monocytogenes
 
is
 
one of 
most dangerous
 
pathogens transmitted through fo
od 
(Stacy et al., 2014) causin
g
 
human illness and death (Painter and Slutsker, 2007)
.
  
The 
growth 
ability of 
L.
 
monocytogenes
 
under refrigerated condition poses
 
a particular concern for
 
ready
-
to
-
eat (RTE) meat products since the
se products are usually kept refrigerated
 
and consumed wit
hout 
reheating (
USDA
-
FSIS
, 2014).  Consequently, 
robust
 
methods to control the pathogen 
in RTE 
meat products are needed to e
nsure the 
safety
 
of 
RTE consumers
.
 
 
1.1.1
 
Characteristics of 
Listeria monocytogenes
   
 
                
 
L.
 
monocytogenes
 
is a Gram
-
p
ositive, short rod
-
shaped, 
non
-
spore forming, facultative
 
anaerobic bacteria 
(
Bell and Kyriakides, 2005)
.
  
L.
 
monocytogenes
 
is a major foodborne human 
pathogen and one of eight
 
important species 
(
L. monocytogenes, L. ivanovii, L. seeligeri, 
L.innocua, L.
we
lshimeri, L. marthii, 
L. grayi
, and L. rocourtiae
)
 
currently known within the 
genus 
Listeria
 
(Rocourt and Buchrieser, 2007)
.
 
 
At present, 
13 serotypes
 
of 
Listeria
 
are
 
known
, 
with the
 
s
erotypes 1/2a, 1/2b, and 4b 
causing most
 
foodborne 
infections in humans 
(CDC, 2013
).  
Listeria 
can grow and survive 
at 
temperature
s of
 
0
-
42
°
C, pH 4.0
-
9.6, 
a
w
 
0.90, and 
10% NaCl 
(
Adam and Moss, 2008
; 
Bell and Kyriakides, 2005; Sauders and Wei
dmann, 2007
). 
 
The concern 
of this organism in foods is its psychrotrophic characterist
ics, acid and salt tolerance, a
nd 
ubiquitous presence in nature
 
including
 
food processing plants
 
and home
-
kitchen (Cox et al
.
, 
1989).
  
Although 
L.
 
monocytogenes
 
is eliminated during normal 
cooking
, it can 
contaminate 
foods 
during post
-
thermal 
processing
 
su
ch as peeling, slicing, and
 
re
-
packaging
.  
Thus, it is not 
6
 
 
surprising that s
everal 
intervention 
strategies have been 
evaluated and 
implemente
d
 
to control
 
L.
 
monocytogenes
 
in food products
.
 
 
1.1.2
 
Listeriosis
 
and manifestation
 
 
 
L.
 
monocytogenes 
is responsi
ble for 
an infect
ious disease
 
called listeriosis 
(Painter and 
Slutsker, 2007)
.
  
The organism is typically transmitted 
via contaminated 
food (Norton and 
Braden, 2007)
.  Listeriosis
 
frequently occur
s
 
in susceptible persons such as neonates, 
the 
elderly, 
preg
nant women, a
nd immunocompromised person
s.  Symptoms 
of listeriosis 
include fever, 
vomiting
,
 
and diarrhea
, which
 
can lead to septicemia, meningitis, and central nervous system 
infections.  The infection is severe in pregnant women and can result in spontan
eous abortion, 
premature birth, and death of the infant (Bell and Kyriakides, 2005
; 
Fsihi et al., 2001
; 
Painter 
and Slutsker, 2007
).
  
Although the number
s
 
of listeriosis cases are not high compared to other 
foodborne pathogen
s
, 
L.
 
monocytogenes 
ranks 2
nd
 
a
mong 
other foodborne pathogens 
in terms of 
fatalities 
and 
induces 
an 
annual loss of 
$2.6 billion due to illness 
in the United States 
(Scallan et 
al., 2011; Hoffmann et al., 2012). 
 
These 
concerns
 
underline the importance of controlling 
this
 
pathogen 
in foo
d
s.
 
 
1.1.3
 
Listeriosis 
outbreaks and incidence
s
 
of 
Listeria monocytogenes 
in RTE meat 
products
 
L.
 
monocytogenes
 
was recognized as a foodborne pathogen in 1981, 
wh
en it
 
was 
linked 
to consumption of contaminated coleslaw in Canada (Schlech et al., 1983).  Th
e first listeriosis 
outbreak in
 
the
 
U.S. 
occurred after consuming
 
pasteurized milk in 1983 (Fleming et. al., 1985).  
After that,
 
listeriosis
 
ha
s
 
been continuously reported from a variety of food products such as 
7
 
 
cheese, milk, pr
ocessed meat, and fresh prod
uce
 
including
 
sprouts, celery
,
 
and cantaloupe 
(Cartwright et al., 2003; Norton and Braden, 2007
;
 
CDC, 2014b
)
.  Th
e first listeriosis outbreak 
from 
processed meat (hot dog) was 
occur
r
ed
 
in 
the 
U.S. in 1988
,
 
with
 
the 
numerous 
reported 
outbreaks 
from con
sumpt
ion of processed meats
,
 
thereafter 
(
CDC, 2014b
).  In 
1998,
 
a multistate 
outbreak of listeriosis in which hot dogs 
manufactured by Bil Mar Foods, Michigan 
were 
implicated, resulted in 101 hospitalizations, 15 deaths, and 6 stillbirths or miscarriages, and 
r
ecalls 
of 
35 million pounds of hot dogs and deli meats (CDC, 1999).
  
In 2000,
 
a multistate 
outbreak of listeriosis associated to the c
onsumption of slice 
deli meat
 
manufactured by
 
Cargill 
Turkey Products, Inc., Texas
 
resulted in 29 cases with 4 deaths and 
3 miscarriages, 
and 
recalls
 
of
 
processed turkey
 
and chicken deli meat
s
 
from the
 
company
 
(CDC, 2000)
.
  
In 2002, a multistate 
outbreak of listeriosis 
linked to the consumption of slice turkey deli meat
 
manufactured by 
Pilgrim's Pride Foods
, 
Pennsylvania
 
resu
lted in 
46 cases with 7 deaths and 3
 
stillbirths or 
miscarriages, 
and 
recalls
 
of
 
27.4 million pounds
 
of fresh and frozen ready
-
to
-
eat turkey and 
chicken products 
(CDC, 2002)
.
  
The foodborne outbreaks associated to the consumption of RTE 
meat products conta
minated with 
L. monocytogenes 
are summarized in Table 1.1.  Several 
outbreaks of listeriosis associated to the consumption of RTE meat products
 
led
 
the USDA
-
FSIS 
to 
issue their
 
interim final rule (
Listeria
 
Rule) for the control of 
L. monocytogenes 
in RTE m
eat 
and poultry products (
USDA
-
FSIS
, 2003
a
).
 
 
 
 
 
 
8
 
 
Table 1.1  Listeriosis outbreaks in the U.S. associated with RTE meat products (CDC, 2014
b
).
 
Year
 
State
 
Food Vehicle
 
No. of Hospitalization
 
No. of Death
 
1998
 
1998
 
1999
 
1999
 
1999
 
1999
 
2000
 
2002
 
2005
 
2006
 
Co
lorado
 
Multistate
 
Florida
 
New York
 
Minnesota
 
Multistate
 
Multistate
 
Multistate
 
Multistate
 
Ohio
 
Hot dog
 
Hot dog
 
Deli meat
 
Hot dog
 
Deli meat
 
Pate
 
Deli meat (sliced turkey)
 
Deli meat (sliced turkey)
 
Deli meat (sliced turkey)
 
Ham
 
4
 
101
 
2
 
4
 
5
 
11
 
29
 
46
 
13
 
3
 
-
 
21
 
1
 
-
 
1
 
-
 
7
 
10
 
1
 
-
 
 
Many major 
listeriosis 
outbreaks have 
been 
traced to 
the 
consumption of RTE meat
s
 
contaminated
 
after thermal 
processing 
(Cartwright et al., 2013)
.  
Among various food products, 
meat products
 
such as 
RTE meats, frankfurters, deli meat, 
an
d 
pate
 
have been ranked first
 
as 
the 
types of food vehicles involved in listeriosis outbreaks due to the extended storage and frequent 
consumption with no additional heat
ing
 
(Cartwright et al., 2013; FDA/CFSAN and 
USDA
-
FSIS, 
2003b
; Farber et al., 2007).
   
 
 
 
 
Up to 2013
, the incidence of 
listeriosis 
had
 
not changed si
gnificantly since 2006 with 
0.26
 
cases reported per 100
,000 population
, 
91% 
of these cases 
led to hospitalization
,
 
and 19.5
% 
resulted in death 
(
CDC, 2014a
).  From 1999 to present, several RTE m
eat products were 
documented as food vehicles responsible for listeriosis including hot
 
dogs, pate, 
and 
deli meat
-
9
 
 
sliced 
turkey 
(
CDC, 2014b
).  Among those food catego
ries, deli meats and hot dog
s were 
reported to be responsible for 7
0% of listeriosis cases
 
in 
the 
U.S.
 
during 1998
-
2008 (Cartwright 
et a
l., 2013).  These data indicate
 
that processed RTE meat
 
product
 
is
 
the primar
y food vehicle 
for human listeriosis (FDA/CFSAN and 
USDA
-
FSIS, 2003b
).
 
 
1.1.4
 
USDA guidelines to control 
Listeria monocytogenes
 
in RT
E meat products
 
 
The emergence of problem from 
L. monocytogenes 
in processed meat and poultry 

-
FSIS, 2003
a
).  
In 1987, USDA
-
FSIS 
developed a 
monitoring and ve
rification program
 
for 
L. monocytogenes
 
in meat products inc
luding beef jerky, 
roast beef, cooked beef
,
 
cooked corned beef, sliced ham
,
 
luncheon meat, small
-
diameter sausage,
 
large
-
diameter sausage, cooked/
uncured poultry, salads and spreads, and dry and semi
-
dry 
fermented sausage
s
 
(
USDA
-
FSIS, 2014
).  
USDA
-
FSIS als

for 
L. monocytogenes
 
in RTE foods in 1989, indicating that any amount of 
L. monocytogenes 
in 
RTE meat or poultry products renders it adulterated and subject to a voluntary recall (
USDA
-
FSIS
, 2003
a,b
).
  
This program r
esulted in dec
reas
ing the rate of illness from 
L. monocytogenes 
for 44% and the rate of death by 48% 
during 1989
-
1993
 
(
Tappero et al., 1995; 
USDA
-
FSIS, 
2003a)
.
  
 
In 2003, 
USDA
-
FSIS issued an interim final rule (
Listeria
 
Rule) for the control of 
L. 
monocyto
genes 
in RTE meat and poultry products (
USDA
-
FSIS
, 2003
a
). According to the Rule, 
three alternative methods were established to control 
L. monocytogenes
 
contamination in RTE 
products: Alternative 1 
-
 
apply both a post
-
lethality treatment and an antimicrobi
al agent or 
process to suppress growth of 
L. monocytogenes
, Alternative 2 

 
apply either a post
-
lethality 
10
 
 
treatment or antimicrobial agent to control growth of 
L. monocytogenes
, Alternative 3 
-
 
use of 
sanitation control measures to prevent recontamination 
after processing (
USDA
-
FSIS
, 2012).
  
 
In 2004, the report of the assessment of effectiveness of 
L. monocytogenes
 
in
terim final 
rule showed that this rule had positive impact in addressing 
L. monocytogenes 
(USDA
-
FSIS, 
2004
)
.
  
After 10 years since USDA
-
FSIS 
issued the interim final rule
, t
he dat
a 
from 
the USDA
-
FSIS monitoring and sampling program
 
indicated that
 
the percent positive in testing for 
L.monocytogenes 
in RTE products has decreased from 0.76% in 2003 to 0.34% in 2013 (
USDA
-
FSIS, 2015
).
 
 
 
In response
 
to
 
the
 
Listeria 
rule, several post
-
lethality treatments and antimicrobial agents 
to control 
L. monocytogenes
 
have been studied.  For example, Bowman et al. (2008) found that 
the application of high hydrostatic pressure processing (HPP) 
at 
400
-
600 MPa 
prev
ented 
Listeria
 
growth due to 
cell struc
ture and gene
 
damage
.  
Chun et al. (2009) reported that the UV
-
C 
irradiati
on (1000
-
8000 J/m
2
) effectively decreased 
L. monocytogenes
 
populations on RTE sliced 
ham.  Foong et al. (2004) showed that the exposure of vari
ous RTE meat products 
to
 
irradia
tion 
reduced the populations 
of 
L.
 
monocytogenes
 
depending
 
on the dose 
of irradiation
.  
 
 
 
 
 
 
 
 
 
11
 
 
1.2
 
Organic acids 
 
1.2.1 
 
Nature of organic acids
 
Organic acids are the compound
s containing carbon in the structure with acidi
c 
properties.  
They can be found 
as natural constituents or additives
 
in
 
food
s
.  The most common 
functional group of organic acid
s
 
is the carboxyl group (
-
COOH)
, 
however
 
alcohol with
 
a
 
hydroxyl group (
-
OH) and the 
organic c
ompounds containing thiol (
-
SH), 
enol
, or 
phenol group
s
 
are also referred to as organic 
acids.  Organic acids are 
weak
 
acids since they do not fully 
dissociate
 
in water.  The two basic forms of organic acids are pure acid
s
 
such as lactic acid, 
propionic acid, acetic acid, 
and 
benzoic acid
, as well as
 
buffered acid
s
 
whic
h are the calcium or 
sodium salts
 
of pure organic acids (Theron and Lues, 2011).  The buffered form has advantage
s
 
over the pure form since it does not 
significantly 
change the
 
pH of the food, 
is safe to handle
, 
and is
 
less 
corrosive to the mach
ines (Theron and Lues, 2007).  Chemical structures of some 
o
rganic acids are shown in Fig.
 
1
.1
.
 
12
 
 
 
Figure 1
.1
  
Chemical structures of some organic acids frequently used in food.
 
 
 
 
 
Formic acid
 
Aectic acid
 
Propionic acid
 
Lactic acid
 
Malic acid
 
Benzoic acid
 
Sorbic acid
 
Tartaric acid
 
Oxalic acid
 
Citric acid
 
13
 
 
1.2.2
 
Organic acids as antimicrobial agents
    
 
Due to
 
severa
l outbreaks from consumption of
 
RTE meat products
 
contaminated with 
L. 
monocytogenes
, 
various
 
strategies 
have been 
investigate
d and implemented
 
to control 
L. 
monocytogenes
.  The use of antimicrobial agents is one 
method
 
recommended by
 
USDA
-
FSIS 
(
USD
A
-
FSIS
, 2003
b
).  A
n a
ntimicrobial agent is defined as a substance that effectively reduces 
or eliminates microorganisms
 
or 
suppresses
 
growth 
to no more than 2 log units 
throughou
t the 
shelf life of the product
 
(
USDA
-
FSIS
, 2003
b
).  
These
 
antimicrobial subst
ances 
adversely impact 
microbial 
protein synthesis
, 
enzym
e activity
, cell membrane and/or cell wall, 
and/
or transport 
mechanisms for 
nutrients
 
(
Lück and Jager, 1997)
.
  
Organic acids have been widely used as 
antimicrobials 
due to their effectiveness and low
 
cost in minimizing microbial 
growth
 
(Theron 
and Lues, 2007; Mani
-
López
 
et al., 2012).  
 
The undissociated form of 
an organic acid
 
is responsible for antimicrobial activity due to 
its hydrophobicity
, enable penetration
 
through 
the microbial 
cell membrane 
a
nd subsequent
 
inhibitory action
 
(
Lück and Jager, 1997)
.  
Once
 
penetrate
, the organic acids lead
 
to
 
cytoplasmic 
acidification, toxic anion accumulation, and disruption of essential metabolic reactions (Theron 
and Lues, 2
011; Lopez et al., 2012) (Fig
 
1.2).  
 
 
 
 
Figure 
1.
2  Mechanism
 
of 
antimicrobial action of organic acids in
 
a 
microbial cell (Lopez et al., 
2012).
 
 
14
 
 
1.2.3
 
Applications of organic acids to food products
 
Organic acids are applied to a wide variety of food
s
 
including RTE meat products to 
control 
Listeria 
spp.  Several organic acids and 
their application method
s 
have been significantly 
improved 
to control
 
Listeria 
including
 
spra
ying, dipping, 
and 
incorporating into 
product 
formulation, or
 
a
ntimicrobial packaging
 
(Stekelenburg, 2003; Barmpalia et al
., 2004; Uhart et al., 
2004; Stopforth et al., 2010)
.  Moreover, 
several studies showed that 
various
 
organic acid
s
 
in 
combination with 
other 
antimicrobial 
compounds
 
or post
-
let
hality treatment
s are
 
more effective 
than 
a single organic acid
.
  
However, organ
ic acid
s
 
or their 
salts reportedly impar
t 
strong acid 
odor
 
in the product
 
(Blom et al., 1997; Islam et al., 2002; Stekelenburg and Kant
-
Muermans, 
2001).
   
 
According to
 
Blom et al. (1997), the mixture of 2.5% lactate and 0.25% acetate 
inhibited
 
L. monocyto
genes
 
in se
rvela
t and cooked ham throughout 5 weeks of storage at 4
°C
 
but the 
inhibition
 
was not maintained in 
the 
cooked ham after 3 weeks of storage at 
9
°C
.  Additionally
, 
consumer acceptance of servela
t formulated with 
a 
lactate/acetate mixture was less
 
than 
that 
formulated
 
without 
an 
acid mixture due to 
the 
s
our taste related to the 
lactate/acetate mixture. 
 
Bedie et al
.
 
(200
1) reported that 
higher
 
organic acid concentration
s provided
 
better
 
of
 
L. 
monocytogenes 
inhibition
 
on frankfurters
.  
Using
 
3% sodi
um lactate
 
currently allowed by 
USDA
-
FSIS inhibited the growth of 
L. monocytogenes
 
on frankfurters during storage at 4
°C
 
for 
70 days 
and using 0.25% sodium diacetate provided the inhibition for 35
-
50 days.  When
 
using 
twice the concentration of both organi
c acids, they
 
showed the complete inhibition for 120 days
 
at 4
°C
. 
 
 
Islam et al. (2002) found tha
t dipping frankfurters in 
up to 25%
 
sodium benzoate, sodium 
propionate, potassium sorbate, or sodium diacetate 
solution 
alone (yield < 0.3% residue of 
15
 
 
inhibito
r to frankfurter) 
was not sufficient to control 
L. monocytogenes 
growth during abusive 
temperature storage
 
at
 
13 and 22
°C
.  They also reported that the 
flavor and overall acceptability 
scores for 
frankfurters treated with sodi
um diacetate were lower compar
ed to
 
the 
no
n
-
diacetate 
treatments
.
 
Glass et al. (2002) reported that 
treating
 
wi
eners with
 
6% 
lactate or 3% diacetate and 
dipping of wieners into the combined solution 
did not 
delay the growth of 
L. monocytogenes
 
during refrigerated storage
. 
 
While
 
the in
clusion of lactate and diacetate mi
xture in the weiner 
formulation
 
can inhibit the growth of 
L. monocytogenes 
at 4.5
°C
 
s
torage for 60 days, this 
treatment was
 
less ef
fective 
at 7
°C
.
 
According to Ba
r
mpalia et al. (2004),
 
incorporating 
1.8% 
sodium lactate or
 
0.25% 
sodium diacetate into 
a 
frankfurter formulation retarded 
L. monocytogenes
 
growth 
during storage 
at 
10
°C
.  When 
both organic acids 
were used 
in combination 
or combine
d
 
with dipping in either 
2.5% lactic acid or 2.5% ace
tic acid after processing, 
List
eria
 
inhibition was improved. 
 
Hwang and Tamplin (2007) reported a model t
o predict the lag phase and 
growth of 
L. 
monocytogenes 
in ground ham as affected by the level of sodium lactate (1.0 

 
4.2
%) and sodium 
diacetate (0.05
-
0.2%)
 
at various temperatures 
(0
-
45
°C
).  The
 
model showed that 
higher
 
lactate 
and diacetate concentrations extended the lag phase at low (
q
 
15
°C
) but not at high storage 
temperature
s
.
     
 
Organic acids and their salts show significant antilisterial activity and are frequently 
sprayed,
 
dipped, or incorporated into product formulations (Bedie et al., 2001; Glass et al., 2002; 
Samelis et al., 2005).  Moreover, some organic acids have been 
used
 
to create antimicrobial 
packaging 
materials 
(Guo et al., 2014).  Currently, organic acid salts a
re allowed in meat and 
poultry products to inhibit microbial growth, with th
e limit of 4.8%, 0.25%, and 4.8
% for 
16
 
 
potassium lactate, sodium diacetate, and sodium 
lactate, respectively, in product 
formulation
s
 
by 
weight (Code of Federal Regulations, 2011).  
 
 
The antilisterial efficacy of these acid salts is affected by many factors including pH, 
water activity, nitrite, salt content, and storage condition
s
 
(Samelis et al., 2003; Seman et al., 
2002).  Extensive research has been conducted to determine the sur
vival of 
L. monocytogenes
 
in 
RTE meat products by dipping in 
solutions of sodium diacetate or sodium lactate
 
alone (Islam et 
al.
, 2002; 
Uhart et al., 2004).  These acid salts were more effective (Bampalia et al., 2004) when 
used together rather than alone 
(Glass et al., 2002; Samelis et al., 200
2; Stekelenburg, 2003);
 
however,
 
they usually 
impart 
strong acid
ic
 
odor (Blom et al., 1997; Islam et al., 2002; 
Stekelenburg and Kant
-
Muermans, 2001).   
 
 
 
 
 
 
 
 
 
 
 
17
 
 
1.3
 
Hop and hop extracts
 
1.3.1
 
Hop plant
 
 
H
op plant 
(
H
umulus lupulus
) belongs to the family 
Cannabina
ceae
 
and only female hop 
flower
s
 
are used for commercial purposes (Verzele and Keukeleire, 1991).  Germany and 
the 
USA are the two
 
largest hop growers with about
 
70% of the total world production in 2013 
(IH
GC, 2014)
.  The hop plant has long been recognized
 
as 
a 
food 
as early as the first century 
A.D., 
as 
a medicinal additive
 
in 
the
 
8
th
 
and 9
th
 
century
 
(Verzele and Keukeleire, 1991), and 
as 
a 
food ingredient
 
in beer production from the 12
th
 
century (Hass & Ba
rsoumian, 1994; Hoffman, 
1956).
  
Only female hop cones containing small yellow granules called
 
lupulin glands are used to 
flavor and preserve 
beer (Verzele and Keukeleire, 1991; Tim, 2003).  In beer processing, dried 
hops are us
ually added during wort boil
ing. 
 
H
owever, hop extracts 
have 
become
 
more popular 
than the traditionally dried hops due to the convenience, 
uniformity, 
stability, economic cost, and 
good quality (Wilson et al., 2003).  
 
 
 
 
 
 
 
 
 
18
 
 
1.3.2
 
Hop extracts
 
When extracted from hop cones with an 
organic solvent
 
(hexane or ethyl alcohol)
 
or 
carbon dioxide, the resulting fract
ions contain mainly alpha
-
 
and beta
-
acids
,
 
with the remainder 
consisting of oils, waxes and uncharacterize
d resins (Wilson et al., 2011).  The composition of 
hops is shown in T
able 
1.
2
.  The constituents with differing side chain
s
 
for
 
hop alpha
-
acids 
are 
humulone, cohumulone, and adhumulone and those of hops beta
-
acids are lupulone, colupulone, 
and adlupulone 
(Fig.
 
1.3
) 
(FDA 2001; Srinivasan et al, 2004).
  
In beer processing, on
ly hop 
alpha
-
acids undergo the isomerization process during 
the 
wort bo
i
ling stage
 
with the result
 
of
 
iso
-
alpha
-
acids providing 
the 
bitter taste to beer
 
(
Keukeleire, 2000; 
Sakamoto and Konings, 
2003
)
.  In order to
 
maintain
 
the 
desirable level bitterness in
 
beer
, iso
-
alpha
-
acid
s are 
currently 
produced off
-
line to facilitate
 
their 
add
ition 
at any stage during beer processing 
(
Keukeleire, 
2000).  To 
prevent beer off
-
flavor due to the decomposition of iso
-
alpha
-
acids w
hen exposed to 
light, 
hop 
acids are 
reduced
 
and formulated as potassium salts in concentrated aqueous solut
ion
s
 
(Keukeleire, 2000) (Fig.
 
1.3
).  These reduced iso
-
alpha
-
acid compounds 
are stable to light and
 
stabilize beer foam (Tim, 2003).        
 
 
 
 
 
 
 
19
 
 
Table 1.2
 
Composition of hops (Verzele and Ke
ukeleire, 1991).
 
Component
 
% by weight
 
Alpha acids
 
Beta acids
 
Amino acids
 
Cellulose
 
Essential oil
 
Monosaccharides
 
Oils and fatty acids
 
Pectins
 
Polyphenols (Tannins)
 
Proteins
 
Salts (ash)
 
Water
 
2 

 
12
 
1 

 
10
 
0.1
 
40 

 
50
 
0.5 

 
5
 
2
 
Trace to 25%
 
2
 
2 

 
5
 
1
 
10
 
8
 

 
12
 
 
 
 
 
20
 
 
 
 
 
Figure 
1.3
 
 
Some h
op compounds
 
and reduced iso
-
alpha
-
acids
 
(Adapted from Keukeleire, 2000).
 
 
 
 
 
 
 
 
 
 
 
21
 
 
1.3.3
 
Hop
s
 
as antimicrobial agent
s
 
Hop compounds
 
have 
received 
increasing 
attention due to their
 
antimicrobial activity 
against 
beer spoilage mi
croorganisms, especially 
G
ram
-
positive bacteria (
Haas and Barsoumian, 
1994; Bhattacharya et al., 2003
; 
Sakamoto and Konings, 2003
)
. 
 
The main target of hop acids in 
bacterial inhibition is 
the microbial cell membrane
 
(
Teuber 
and Schmalreck, 1973).  
 
In Gra
m
-
negative bacteria, 
however, 
serumphosphatides 
presented 
in 
the 
outer membrane of bacteria cell
s
 
inactivate both lupulones and humulones, and minimize the bactericidal activity of the hop 
components
 
(Teuber and Schmalrek, 1973).  
However, some studies 
hav
e shown that hops in 
combination wi
th other antimicrobial agents are
 
effective in contro
lling some G
ram
-
negative 
bacteria.  Fukao et al. (2000) found that the combination of 
100 ppm 
hop resin
 
with 
0.5% 
sodium 
hexametaphosphate inhibited 
the growth of 
E. co
li 
K
-
12 IFQ3301 in broth and mashed potato
e
s
,
 
whereas using either
 
hop resin
 
or sodium hexametap
hosphate 
alone did not inhibit
 
E. coli
.
  
Natarajan et al. (2008) also showed that lupulone (beta
-
acid) 
in combination 
with 
polymyxin B 
sulfate
 
inhib
ited
 
Proteus
 
vulgaris
, 
Serratia marcescens
,
 
and 
Proteus mirabilis
. 
 
 
Hop acids are weak acids and the inhibition of bacterial growth is mainly attri
buted to the 
undissociated form
 
(
Simpson and Smith, 1992; 
Sakamoto and Konings,
 
2003).  The 
undissociated form
 
of hop 
ac
id
s can invade the microbial cell and dissociate into protons and ho
p 
anions, resulting in lowering of
 
the
 
intracellular pH and diffusion of divalent cations out of the 
cell 
(Fig.
 
1.4
)
 
(Sakamoto and Konings, 2003)
.  
I
nhibition of bacterial growth 
also has 
been 
attribut
ed 
by the prenyl group on
 
the side chain of hop acid which
 
causes
 
cell membrane leakage
 
(
Teuber 
and Schmalreck, 1973; Schmalreck and Teuber, 1975
; 
Keukeleire, 2000)
.  
 
22
 
 
 
Figure 
1.4
 
 
Hop m
echanism
s 
i
n 
a 
microbial cell (Sakamoto and Konings, 200
3).
 
Numerous 
studies have demonstrated the antimicrobial activity 
of hop acids.  Hop 
compounds showed 
inhibit
ion against
 
Bacillus subtilis 
(Schmalreck and Teuber, 1975), fungi 
(Mizobuchi and Sato, 1985), 
Lactobacillus
 
acidophilus 
(Todd et al., 1992), 
Strep
tococcus 
mutans
 
(
Bhattacharya et al.,2003)
, 
protozoa (
Srinivasan et al., 2004), and l
actobacilli (
Rü
ckl
e
 
and Senn, 2005).  
Millis et al. (19
94) showed that 6 ppm beta acid
 
completely inhibited 
L. 
monocytogenes
 
growth 
in brain heart infusion broth after inc
ubation at 35
o
C for 24 h.  L
arson et 
al. (1996) showed that
 
10 

L
 
of hop extract
 
contained
 
41% 
beta acid and 
12% alpha acid 
and 
the 
10 

L
 
of hop extract containing
 
94.7% beta acid 
completely inhibited 
L. monocytogenes
 
in trypticase soy broth and brain heart infusion broth, respectively
,
 
after incubation at 37
o
C 
for 
24 h.  
Th
e antilisterial activity of hop
 
beta acids (1.0
-

mixed with other antimicrobial agents (Shen and Sofos, 2008; Shen et al., 2009). 
 
 
For practical application in RTE meat products, the 
USDA
-
FSIS
 
approved hop 
beta acids 
as generally recognized as safe (GRAS) 
for frankfurter casings and 
cooked 
ready
-
to
-
eat 
meat and 
poultry products 
(US/
FDA GRAS Notice Nr 000063) (
USDA
-
FSIS
, 2013
).  Currently, several 
hops extracts are produced by fractionation and chemical conve

demand (Mahaffee et al, 2009; Wilson et al., 2011).  However, 
almost no studies
 
have been 
23
 
 
conducted to evaluate 
the 
antilisterial activity of various hop extracts
,
 
including isomerized or 
reduced forms 
in
 
liquid media and
 
meat p
roducts
 
with/without organic acid salts
. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24
 
 
CHAPTER 
2
: 
INHIBITION OF 
LISTERIA MONOCYTOGENES
 
IN FULL AND LOW 
SODIUM FRANKFURTERS AT 4, 7, OR
 
10ºC
 
USING SPRAY
-
DRIED MIXTURES OF 
ORGANIC ACIDS SALTS
 
 
2.1
 
Introduction
 
Var
ious 
organic acid salts 
have been used as antimicrobial agents 
singly or jointly 
against 
L. monocytogenes 
in RTE meat products to 
improve the product safety
 
with no
 
qual
i
ty loss.
  
Recently, 
several 
powder form
s
 
of organic salts have been developed as 
Listeria
 
growth 
inhibitors
 
bu
t scientific studies on their efficacy 
of 
inhibition in RTE meat products 
have not 
been conducted enough
. 
 
Hence, t
he purpose of this research 
was
 
to evaluate the 
impact 
of five 
powder
ed 
(
the mixtures of 
sodium 
lactate
, sodium 
acetate
, sodium diacetate, po
tassium acetate
, 
and/or
 
potassium diacetate)
 
and four 
liquid inhibitors (
the mixtures of sodium lactate, sodium 
di
ac
etate,
 
and/or potassium lactate
) 
on 
Listeria
 
inhibition, organoleptic quality, and 
physicochemical properties of frankfurters.  
Th
e hypothes
is of this research i
s 
that the powdered 
organic acid salts exhibit antilisterial activities as same 
as 
or better than the 
current commercial 
inhibitors 
in liquid for
 
full
-
 
and low
-
sodium frankfurters without significant impacts on 
physicochemical and orga
noleptic characteristics. 
 
 
2.2
 
M
aterial
 
and methods
 
T
wo sets of experiments
 
were conducted
. 
Initially, 
10
 
full
-
sodium frankfurter 
formulation
s
 
containing organic acid salt or 
their
 
mixtures 
(5 powdered, 4 liquid and 1 control) 
were assessed for 
Listeria
 
i
nhibition, organoleptic quality, and physicochemical properties
. 
Thereafter, 
five
-
low sodium frankfurters
 
containing three 
best 
powdered
 
inhibitors
, 
one
 
liquid 
control, 
and 
one inhibitor
-
free 
control
 
were 
similarly 
evaluated
.  
 
 
25
 
 
2.2
.1
 
Full sodium f
rankfurt
er preparation 
using
 
powdered or liquid inhibitors
 
 
Full sodium (1.8% salt) frankfurters were manufactured in the Michigan State University 
Meat Laboratory (East Lansing, MI). 
Raw meat (boneless pork butt, pork back fa
t, and 80% 
ground beef) and non
meat in
gredients were purchased locally
, 
and
 
the powdered and 
liquid
 
inhibitors were obtained
 
from 
Niacet
 
b.v. (Tiel, The Netherlands). Both pork butt and back fat 
were coarsely ground 
using a 
0.95 cm plate
 
(Model 4146, Hobart Corporation, Troy, OH)
 
and 
pre
mixed 
for 3 min (Model Butcher Boy 250F, Lasar Mfg C
o
. Inc.,
 
Los Angeles, CA). 
 
In
 
each of three replications, 10
 
different frankfurter formulations (22.
7
 
kg/batch) were 
randomly prepared 
by adding
 
water
 
(no inhibitor)
 
or 
one of the 
inhibitor
s
 
(
powdered or liqui
d
)
 
(Table
s
 
2.1 through
 
2.
3) by weight (w
t
/w
t
) as follows: 
(1
) no inhibitor
 
(water)
 
control: CTR;
 
(2
) 
0.25% sodium lactate (SL) + 
0.25% sodium acetate (SA): powdered inhibitor (
P
I
-
1); (3
) 0.5% 
SL + 0.5% SA: PI
-
2;
 
(4
) 0.2
5
% SL + 
0.2
5
% SA + 
0.16
%
 
sodium diace
tate (SD): PI
-
3; (5
) 0.6% 
potassium acetate (PA) + 
0.15% p
ot
assium diacetate (PD): PI
-
4; (6
) 0.8% PA + 
0.2% PD
: PI
-
5; 
(7
) 1.5% SL + 
1.0% water: 
l
iquid inhibitor (LI
-
1);
 
(8
) 1.4% SL +
 
0.1% SD + 1.0% water: LI
-
2; 
(9
) 1.5% potassium lactate
 
(PL) + 1.0% water:
 
LI
-
3; and (10
) 1.4% PL + 0.1% SD + 
1.0% water: 
LI
-
4. The inhibitor weight difference
s
, due to different amount
s
 
in organic salts and liquid, w
ere
 
adjusted with pork meat and water, respectively.  
 
 
Meat batter prepared
 
on a different day
 
for each 
replicat
ion
 
was blended with 
spice
s
 
and 
preservative
s
 
in a bowl chopper (m
odel K64
-
Va, Maschinenfabrik Seydelmann KG, Aalen, 
Germany) to a final batch temperature 
of
 
12
o
C. The resulting emulsion was then stuffed 
into 
cellulose casing (24 mm, Viscofan USA, Inc., Mo
ntgomery, A
L
)
,
 
which was 
linked
 
(m
odel 500, 
VEMAG Maschinenbau GmbH, Verden, Germany)
 
into 
9
-
 
to 10
-
cm
 
length  and cooked to an 
internal temperature of 70
o
C in a 
smoke
-
free 
smokehouse (m
odel A28, CGI Processing Equip. 
26
 
 
Cicero, IL)
.
 
 
T
he end
-
cook
ing
 
temperat
ure 
was 
confirmed 
with
 
a calibrat
ed
 
digital thermometer
 
and 
logger (
m
odel 800024, Sper Scientific Ltd., Scottsdale, AZ)
. To asse
ss
 
cooking yield, 
four
 
frankfurter links per treatment (10 frankfurters per link) were randomly selected, labeled, and 
weighed b
efore cooking
.
 
After cooking
,
 
rins
ing
 
and surface drying
, the labeled frankfurters were 
re
weighed
 
to determine cooking yield
. The remaining frankfurters 
were 
stored overnight
 
at 
2
o
C
,
 
manually peeled
,
 
placed
 
in pouch
es (p
roduct # 75001979, Koch 
S
upplies Inc
, Kansas 
C
ity, MO)
, 
and vacuum
 
packed 
(Multivac Sepp Haggenmueller GmbH & Co. KG., Wolfertschwenden, 
Germa
n
y). Three groups of samples were prepared for physicochemical, microbial, and 
consumer sensory 
analyses
. 
 
 
2
.2
.2
 
Low sodium
 
frankfurter preparation w
ith
 
powdered or liquid inhibitor
s
 
 
Low sodium 
frankfurters 
(1% salt) 
were 
manufactured as pr
eviously described for the 
full
-
sodium frankfurter 
except using 
the 
following
 
five formulations: 
(1
) no 
i
nhibitor
 
(water)
 
control (
CTR
);
 
(2
) 
0.5% SL
 
+ 
0.5% SA
:
 
PI
-
2
;
 
(3
) 
0.247% SL
 
+ 
0.247% SA
 
+ 
0.156% SD
:
 
PI
-
3
;
 
(4
) 
0.8% PA
 
+ 
0.2% PD
: 
PI
-
5
;
 
and  
(5
) 
1.4
% 
PL
 
+ 
0.1% SD
 
+ 
1.0% water
: 
LI
-
4 
(Table
s
 
2.1 
through 2.
3
). 
 
 
 
 
 
 
 
 
27
 
 
Table 2.1
 
 
Base f
ormulation of frankfurters
.
 
 

 

 

 

 






 





 

 






 

 
 


 





 



 

 



 





 

 



 

 

 




 

 

 

 
 











 







 






 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
28
 
 




 

added 
to frankfurter formulations
.
 
 

 



 

 

 

 

 

 

 

 

 

 

 

 

 


 


 

 

 
29
 
 
Table 2.3
 
 
Percent
 
of powdered inhibitors (PI) and liquid inhibitors (LI) 
added 
to frankfurter formulations
.
 
 
Inhibitors
 
Sodium
 
lactate
 
Sodium
 
acetate
 
Sodium 
diacetate
 
Potassium
 
acetate
 
Potassium
 
lac
t
ate
 
Potassium
 
diacetat
e
 
Water
 
Total
 
amount
 
Control
 
0
 
0
 
0
 
0
 
0
 
0
 
2.5
 
2.5
 
PI
-
1
 
0.25
 
0.25
 
 
 
 
 
 
0.5
 
PI
-
2
 
0.5
 
0.5
 
 
 
 
 
 
1.0
 
PI
-
3
 
0.2
5
 
0.2
5
 
    
0.1
5
 
 
 
 
 
0.6
5
 
PI
-
4
 
 
 
 
0.6
 
 
   
0.15
 
 
0.75
 
PI
-
5
 
 
 
 
0.8
 
 
 
0.2
 
 
1.0
 
LI
-
1
 
1.5
 
 
 
 
 
 
1.0
 
2.5
 
LI
-
2
 
1.4
 
 
 
0.1
 
 
 
 
1.0
 
2.5
 
LI
-
3
 
 
 
 
 
1.5
 
 
1.0
 
2.5
 
LI
-
4
 
 
 
 
0.1
 
 
1.4
 
 
1.0
 
2.5
 
 
 
 
 
 
 
 
 
 
30
 
 
2.2
.3
 
Physicochemical analys
i
s 
 
Seven 
physicochemical p
arameters
 
were 
assessed
: 
pH, 
protein, fat, moisture, 
water 
activity (
a
w
)
, s
odium, and cooking yield.
 
 
For
 
pH
, a 5
-
g sample was homogenized in 25 ml of 
deionized 
water, and the pH
 
was measured 
with a 
meter (Accumet AR15, Fisher Scientific Inc., 
Pittsburgh, PA) 
equipped with a pH 
electrode (m
odel 13
-
620
-
631, Fisher Scientific Inc., 
Houston, TX)
.
 
 
P
rotein, fat, and moisture
 
contents were determined
 
with
 
a 
nitrogen an
alyzer 
(
model 
FP
-
2000 Nitrogen Analyzer, Leco Corp
.
 
S
t
. Joseph, MI),
 
fat extractor (Soxtec System 
HT6, Tecator AB, 
Höganäs
, Sweden), and dry
ing
 
oven (
model 
Yamato DX 400, Yamato 
Scientific. Ltd.,
 
Tokyo, Japan), respectively
,
 
according to AOAC International
 
(
2005
)
 
met
hods 
992.15, 991.36 and 950.46B, 
respectively
.  The 
a
w
 
was determined with
 
an AquaLab meter 
(Decagon Devices, Inc., Pullman, WA). Sodium content was 
determined
 
with a pH
-
ion analyzer 
(m
odel 123, Omnion, Inc., Rockland, MA) 
equipped 
with a sodium
-
specific
 
electrode
 
(
m
odel 
A230T, Omnion, Inc., Rockland, MA)
 
calibrated with standard sodium solutions. Cooking yield 
for each frankfurter formulation 
was 
based on
 
the weight difference before and after cooking
 
in 
the smokehouse
.
 
 
2.2
.4
 
Listeria 
monocytog
enes
 
strains and frankfurter inoculation 
 
The following s
ix 
L. monocytogenes
 
strains
 
of different 
pulsed
-
field gel electrophoresis 
types were selected for use:
 
Lm
-
10
-
s11 (
serotype 
1/2a
, 
delicatessen
 
isolate
), Lm
-
12
-
s11 
(
serotype 
1/2b, 
delicatessen
 
isolate)
, Lm
-
12
-
s8 (
serotype
 
1/2b, delicatessen
 
isolate
), R3
-
031 
(
serotype 
1/2a, food isolate from a hot dog outbreak), N1
-
227 (
serotype
 
4b, food isolate from a 
deli meat outbreak), and R2
-
763 (
serotype 
4b, food isolate from a deli meat outbreak)
, all of 
which wer
e obtained from Dr. Martin Wiedmann (Cornell University, Ithaca, NY)
. Each strain 
31
 
 
had been 
preserved at 
-
80
o
C in Trypticase Soy Broth
 
(TSB)
 
containing 0.6% 
(w/v) 
yeast extract 
(
YE) (Difco, Becton Dickinson, Sparks, M
D
) and 20% glycerin
.
 
 
For the experiment
s, strains 
were
 
subjected to two consecutive
 
cultures in TSB
YE for 24 h at 
37
°
C
,
 
pelleted by 
centrifugation at 
3,100 x g
 
for 15 min at 4
°
C, and then re
suspended in sterile
 
phosphate buffered 
saline (PBS; pH 7.4). The o
ptical density (OD) 
of each cell suspe
nsion 
was measured at 600 nm
, 
suspensions were 
adjusted to 
the same
 
OD value
, and 
 
5 mL
 
of each suspension 
was added
 
to 3 
liters of PBS to obtain a six
-
strain 
L. monocytogenes
 
cocktail containing ~1 x 10
6
 
CFU/mL
. 
The 
L. monocyt
o
genes
 
population 
in 
the 
inoc
ulum was confirmed 
by
 
plating appropriate dilution
s
 
on 
Trypticase soy agar
 
(TSA)
 
(Difco, BD) with YE
 
and incubating 
22 to 24 h 
at 37
°
C.
 
Fifty frankfurters from each 
formulation
 
were aseptically transferred to a mesh bag
, 
immersed in
 
the 
six
-
strain 
L.
 
monoc
ytogenes
 
cocktail, and gently stirred for 1 min. T
he mesh bag 
with the frankfurters was then 
removed
,
 
drained for 
1
 
min, 
and placed
 
in
 
a 
bio
safety hood
 
for 25 
min for 
the inoculum 
to absorb. T
wo
 
frankfurters were aseptically transferred to 
each of 
10
 
Shanv
ac vacuum bag
s
 
(10 by 15 cm outside dimensions; Shannon Packaging, Chino, CA)
,
 
vacuum sealed
, and 
stored at 4, 7, 
and 
10
°
C for up to 90 day
s.
 
Uninoculated frankfurters from 
each 
formulation
 
were 
used to quantify the
 
background bacteria. 
 
 
2.
2
.5   
 
Microbia
l analysis
 
 
Immediately
 
after packaging and 
after 15, 30, 45, 60, 75 and 90 days of storage at 
4, 7
,
 
and 
10
°
C, 
one 
inoculated
 
and one uninoculated 
bag per treatment 
was
 
randomly selected 
to 
quantify 
L
. 
monocytogenes
 
and mesophilic aerobic bacteria
 
(MAB), r
espectively
. 
 
All samples 
(25 g) were diluted 
1:10 in
 
PBS and homogenized
 
in a
 
stomach
er
 
(NEUTEC Group Inc, 
Farmingdale, NY) 
for 1 min. Appropriate serial diluti
ons in PBS were then plated on m
odified 
32
 
 
Oxford a
gar (Difco, M
D
) and TSA
YE to enumerate 
L. monoc
ytogenes 
and
 
MAB, respectively, 
after 48 h of incubation at 35°C.
 
 
The methods for physicochemical analyses, 
Listeria 
inoculation, and quantification of 
Listeria 
and mesophilic aerobic bacteria (MAB) were the same 
as used 
for both full
-
sodium and 
low
-
sodiu
m frankfurters.
 
 
2.2
.6
 
Consumer s
ensory analysis
, full
-
sodium frankfurters
 
 
For sensory analysis of full sodium frankfurters,
 
the following 
two sets of 
samples
 
were 
manufactured in 
three
 
separate batches for evaluation on separate days
: (1
) four
 
sodium
-
bas
ed 
frankfurter
 
formulations 
with 
a 
control
,
 
and (2
) four potassium
-
based frankfurter
 
formulations
 
with 
a 
control. 
F
rankfurters 
containing
 
the 
1% 
powdered mixture of 
0.5% SL 
and 
0.5% 
SA 
were
 
not included 
because
 
the USDA 
does 
not permit 
SA concentrations
 
ab
ove
 
0.25%
 
(US
-
FDA, 
2011)
.
 
A total of 330 frank
furter consumers (110 consumers
 
per 
replication) were recruited from 
student
s, staff, and faculty members at Michigan State University to evaluate the full sodium 
frankfurter formu
lations on three different day
s
 
(
55 panelists each for 
the 
sodium
-
 
and 
potassium
-
based 
formulations
 
plus
 
the 
control
s
)
.
 
 
On the day of evaluation, 
20
 
frankfurters 
of each formulation
 
were gently 
heated with 
agitat
ion
 
in 
separate
 
pots 
of boiling water 
to achieve an internal temperature 
of 72
o
C. The heated 
frankfurters were then 
placed
 
in
 
sealable bag
s, which 
were
 
immersed
 
in 63
o
C water 
until given to 
the panelists. 
These boiling and warming procedures were repeated until 
the 2
-
 
to 2.5
-
h
 
sensory 
evaluation was 
finished
. 
Upon serving, each
 
frankfurter was cross
-
cut 
to a length of
 
4 cm
,
 
placed 
in a 
randomly coded 
4
-
oz 
(120
-
ml) 
soufflé cup
,
 
and covered
. Trays containing
 
samples of 
five 
different 
formulations along with a glass of filtered water were r
andomly presented to each 
33
 
 
panelist
 
in indi
vidual booths equipped with a touch
-
screen computer and controlled lighting
.
 
All
 
samples 
were evaluated 
for appearance, texture, flavor, and overall acceptability 
using
 
a 9
-
point 
hedonic scale (9 = like extremely and 1 = dislike extremely). 
Data
 
were colle
cted using 
the 
Sensory
 
Information Management Systems
 
(
Sensory Computer Systems, Morristown, NJ)
 
a
nd 
included
 
any written comments concerning the samples.
 
 
2.2
.7
 
Consumer s
ensory analysis
, low
-
sodium frankfurters
 
 
For sensory analysis of low
-
sodium frankfu
rters, 
210 frank
furter consumers (105 
consumers
 
per 
replication) were similarly recruited with 20
 
frankfurters per treatment 
prepared 
as previously described
 
in full
-
sodium frankfurters. O
n the day of evaluation
 
for 
appearance
, 
texture, flavor, and overall
 
acceptability
, 
trays
 
containing
 
samples 
of four
 
different 
formulations 
along with a glass of filtered water were 
randomly presented 
as previously described.  
 
 
2.2
.8
    
Statistical analysis 
 
P
hysicochemical
 
and microbial 
d
ata were subjected to 
the
 
general
 
linear model procedure 
of SAS
 
(SAS Institute, 2002)
.
 
 
To better assess the effect of treatment on 
Listeria 
inhibition, the 
area under the
 
graph of 
Listeria 
population in frankfurters 
during storage 
fo
r each treatment was 
calculated
.
 
 
The higher area under
 
graph, the better growth of 
L. monocytogenes.
  
Means
 
were 
compar

a
 
= 0.05 
level. 
 
For sensory analysis
, a 
mixed model 
analysis of 
variance
 
was used
 
for
 
comparison
 
of means with 
Tukey

 
Test
 
at 
a
 
= 0.05.
 
 
 
 
 
34
 
 
2.3
 
    
 
R
esults and disc
ussion
 
2.3
.1
 
Physicochemical analysis
 
 
Ten full
-
sodium 
frankfurter formulations 
(CTR, PI
-
1 t
hrough
 
PI
-
5, and LI
-
1 t
hrough
 
LI
-
4) and five low
-
sodium frankfurter formulations (CTR, PI
-
2, PI
-
3, PI
-
5, and LI
-
4) were assessed 
for seven physicochemical parameter
s: 
sodium, pH, a
w
, cook yield, moisture, protein, and fat
 
(Tables 
2
.
4 and 
2
.
5).
 
 
Sodium 
concentration
s 
were 
correlated
 
with the amount of sodium present 
in the 
Listeria 
inhibitors. 
 
F
ull sodium f
rankfurter formulations PI
-
2
, 
LI
-
1,
 
and LI
-
2 were highest 
in 
sodium (1
,
279 

 
1
,
345 mg/100g
 
of sample
), followed by PI
-
1, 
PI
-
3 (1
,
172
-
1
,178 mg/100g), 
and 
PI
-
4, 5
,
 
LI
-
3, 
LI
-
4
, and CTR (950 

 
1
,035 mg/100g), and 
 
none of 
these formulations
 
contained sodium
-
based 
Listeria
 
inhibitors except 
for LI
-
4 (
0.1% 
SD
). 
 
Similarly
, low
-
sodium 
frankfurter
 
formulation PI
-
2 was higher in sodium
 
(
P 
< 0.05) (926 mg/100g) than PI
-
5
, LI
-
4, and 
CTR
 
(750 to
 
763 mg/100g)
,
 
and
 
PI
-
3 
was
 
intermediate (839 mg/100g). 
 
F
rankfurter
 
pH
 
was influenced by the presence and amount of 
SA
 
(pH 8.9), 
SL
 
(pH
 
6.3) 
and
 
SD
 
(pH 4.5 

 
5.0) in the formulation. 
Full
-
sodium frankfurters containing none or trace (

 
0.1%) amounts of diacetate (PI
-
1, PI
-
2
, 
LI
-
1, LI
-
3
, 
and LI
-
4) were significantly less acidic 
(pH 
6.39
 
-
 
6.41)
 
than those formulations containing diacetate (PI
-
3, PI
-
4, PI
-
5, 
and 
LI
-
2, pH 6.21
-
6.
26
, 
P
 
< 0.05), 
and CTR had
 
an 
intermediate
 
pH of 6.35.  For 
low
-
sodium frankfurters, 
formulations 
PI
-
2, 
LI
-
4, and CTR
 
were less acidic (pH 6.29 

 
6.31)
 
than 
the remaining 
two 
formulations
 
(PI
-
3
 
and
 
PI
-
5
;
 
pH 6.13 to 6.15
;
 
P
 
< 0.05) 
which
 
contained > 0.156% 
PD
. 
 
Similar 
to
 
these findings, Pal et al
. (2008)
 
found
 
that
 
the pH of frankfurters decreased from 6.17 to 6.02 
as the 
SD
 
and 
PL concentrations
 
increased. 
 
Fat and moisture 
content
 
in full
-
 
and low
-
frankfurters were within 2% 
and
 
the remaining parameters (a
w
, cooking yield, 
and 
protein) 
differ
ed by <
1% 
(Table
s
 
2.
4 
and 
2.
5
)
.
 
35
 
 
Table 2
.4
  
Impact
1
 
of 
formulations containing 
powdered and liquid inhibitors
2
 
on 
the 
physicochemical properties 
of
 
full 
 
      
sodium 
frankfurters
.
 
 
Parameters
3
          
 
CTR 
(
0%
)
4
 
PI
-
1 
(
0.5%
)
 
PI
-
2 
(
1.00%
)
 
PI
-
3 
(
0.65%
)
 
PI
-
4 
(
0.75%
) 
 
PI
-
5 
(
1.00%
)
 
 
LI
-
1 
(
2.50%
) 
 
LI
-
2 
(
2.50%
)
 
LI
-
3 
(
2.50%
) 
 
LI
-
4 
(
2.50%
)
 
Standard
 
Error
 
 
              
 
 
 
 
 
 
 
 
 
 
 
S
odium
 
1031
c
 
1178
b
 
1279
ab
 
1172
b
 
1035
c
 
1021
c
 
1345
a
 
1328
a
 
950
c
 
992
c
 
17.93
 
(mg/100g)
 
 
 
 
 
 
 
 
 
 
 
 
 
pH
 
6.35
c
 
6.39
b
 
6.41
a
 
6.21
e
 
6.26 
d
 
6.22
e
 
6.39
ab
 
6.26
d
 
6.40
ab
 
6
.40
ab
 
0.004
 
 
 
 
 
 
 
 
 
 
 
 
 
a
w
 
0.956
a
 
0.955
a
 
0.950
a
 
0.956
a
 
0.950
a
 
0.955
a
 
0.951
a
 
0.949
a
 
0.951
a
 
0.953
a
 
0.002
 
 
 
 
 
 
 
 
 
 
 
 
 
C
ooking
 
yield (%)
 
89.50
b
 
88.51
c
 
90.03
ab
 
90.69
a
 
88.07
c
 
90.08
ab
 
88.03
c
 
88.01
c
 
90.16
ab
 
90.04
ab
 
0.12
 
 
 
 
 
 
 
 
 
 
 
 
 
M
oisture (%)
 
60.79
ab
 
60.02
de
 
60.50
abc
 
60.61
ab
 
60.50
abcd
 
60.84
a
 
60.07
cde
 
59.71
e
 
60.33
bcd
 
60.11
cde
 
0.1
0
 
      
 
 
 
 
 
 
 
 
 
 
 
 
P
rotein (%)
 
14.86
ab
 
15.15
a
 
14.44
bc
 
14.58
bc
 
14.62
abc
 
14.48
bc
 
14.18
c
 
14.65
abc
 
14.32
c
 
14.16
c
 
0.2
0
 
 
 
 
 
 
 
 
 
 
 
 
 
F
at (%)
 
18.67
ab
 
18.59
ab
 
18.84
a
 
18.47
ab
 
17.78
bc
 
18.8
2
a
 
17.03
cd
 
17.42
cd
 
17.71
bc
 
16.55
d
 
0.21
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1
Mean values with 
same letters in the same row were
 
not 
significantly different (
P
 

 
0.05). 
     
 
2
Inhibitors 
in the formulation
 
as in Table 
2.
3
.
 
3
Least square means of 
n
 
= 9
 
to 
36 observ
ations. 
 
4
Amount of inhibitor
.
 
 
 
 
 
36
 
 
Table 
2
.
5
 
 
Impact
1
 
of 
formulations containing 
powdered and liquid inhibitors
2
 
on 
the 
 
      
physicochemical 
properties 
of
 
low sodium 
frankfurters
.
 
 
          
Parameters
3
 
CTR 
(
0%
)
4
 
PI
-
2 
(
1.00%
)
 
PI
-
3 
(
0.65%
)
 
PI
-
5 
(
1.00%
) 
 
LI
-
4 
(
2.50%
)
 
Standard
 
Error
 
 
 
             
 
 
 
 
 
 
 
 
 
 
 
 
 
 
S
odium
 
(mg/100g)
 
750
b
 
926
a
 
839
ab
 
754.89
b
 
763
b
 
19
 
 
 
 
 
 
 
 
 
pH
 
6.3
0
a
 
6.
3
1
a
 
6.
15
b
 
6.
13
b
 
6.
29
a
 
0.0
2
 
 
 
 
 
 
 
 
a
w
 
0.9
71
a
 
0.950
a
 
0.956
a
 
0.955
a
 
0.9
66
a
 
0.002
 
 
 
 
 
 
 
 
C
ooking
 
yield (%)
 
89.5
7
b
 
90.03
ab
 
90.6
9
a
 
90.08
ab
 
89.65
a
 
0.
4
0
 
 
 
 
 
 
 
 
M
oisture (%)
 
6
2.74
ab
 
60.50
abc
 
60.61
ab
 
60.84
a
 
6
2.09
a
 
0.
36
 
      
 
 
 
 
 
 
 
P
rotein (%)
 
14.
37
ab
 
14.44
bc
 
14.58
bc
 
14.48
bc
 
14.
35
a
 
0.
14
 
 
 
 
 
 
 
 
F
at (%)
 
18.
06
ab
 
18.84
a
 
18.47
ab
 
18.82
a
 
1
8.12
a
 
0.
34
 
   
 
 
 
 
 
 
 
1
Mean values with 
same le
tters in the same row were
 
not 
significantly different (
P
 

 
0.05). 
2
Inhibitors 
in the formulation
 
as in Table 
2.
3
.
 
3
Least square means of 
n
 
= 9 
to
 
36 observations. 
 
4
Amount of inhibitor
.
 
 
2.3
.2
 
Microbial growth
 
 
 
Dip inoculati
on
 
yielded average
 
L. monocyto
genes 
population
s
 
of 4.
6
 
and
 
4.
7
 
log CFU/g 
of 
sample 
in full
-
 
and low sodium frankfurters, respectively 
(
Fig.
 
2
.
1
 
and 
2
.
2
).
 
 
After 
storing the 
vacuum
-
pack
ed full
-
sodium frankfurters 
at 4
o
C for up to 90 days, 
all formulations showed better 
Listeria 
inhibiti
on than the 
inhibitor
-
free 
CTR (Fig 2.1 and Table A.2).  
Listeria
 
populations in 
the four
 
diacetate
-
containing formulations (PI
-
3, 
PI
-
4, 
PI
-
5,
 
and
 
LI
-
2
) continuously 
decreased to
 
4.02 (PI
-
4)
, and
 
to 
4.
23
 
(LI
-
2
) 
log CFU/g at 
the 
end of storage
 
(Fig. 2.1 and
 
Table A.1).  
In low 
sodium frankfurters, PI
-
5
 
was the only formulation 
in which 
Listeria
 
decreas
ed
 
to 4.15 log 
CFU/g after 90 days of storage at 4
o
C
 
(Fig. 2.2 and Table A.3)
.  Full sodium PI
-
2
, L
I
-
4 and low
-
37
 
 
sodium PI
-
3 formulations
 
suppressed 
Listeria
 
gro
wth 
at
 
4
o
C for 45
 
and 30
 
days
, respectively,
 
w
hile
 
low
-
sodium PI
-
2 and full
-
sodium
 
PI
-
1 (containing half the inhibitor 
concentration
s found 
in PI
-
2) allow
ed
 
continuous
 
growth
 
to 6.51 and 6.90 log CFU/g, respectively, by the end of 
storage
.
 
 
Two single
 
orga
nic salt formulations (LI
-
1
 
and LI
-
3) extended 
the initial 
Listeria
 
lag 
phase 
in full
-
sodium frankfurters but then allowed
 
populations 
to increase by 
1.
2
 
to
 
1.4 log 
CFU/g 
during
 
storage at 4
o
C
,
 
wh
ereas
 
addition of SD to the same formulations 
either 
decreas
ed 
Listeria
 
populations by 
0.
33
 
log CFU/g
 
or maintained the initial level with almost no growth
, 
respectively
.
 
 
These results agree with several other 
studies
 
in which a 
greater combin
ed
 
efficacy 
was found 
for
 
SD 
than for SL, and SD plus SL
 
was the
 
most ef
fective 
combination for
 
inhibiting 
growth of 
Listeria
 
(Barmpalia et al., 2004; Glass et al., 2002; Mbandi and Shelef, 2001; Schlyter 
et al., 1993; Stekelenburg, 2003)
.  
 
Listeria
 
populations in the 
low
-
 
and full
-
sodium
 
inhibitor
-
free 
CTR
 
exceed
ed
 
7.
0
 
log 
C
FU/g after 
30 
to
 
45 days of storage at 4
o
C, and 
15 to
 
30 
days 
of storage 
at 7 and 10
o
C
, 
respectively
.
 
 
O
f 
the 
10
 
full
-
sodium formulations
, only PI
-
4
 
and PI
-
5 
had
 
listericidal activity (
-
0.
54
 
to 
-
0.
55
 
log CFU/g) at 7
o
C during 90 day
s of
 
storage
, 
whereas 
PI
-
5 in low
-
sodium 
frankfurters was listericidal (
-
0.
24
 
log CFU/g) at 7
o
C and listeristatic (+0.
02
 
log CFU/g) at 10
o
C 
during storage. 
 
Listeria
 
inhibition also 
was reported by Barmpalia et al. (2004)
 
when 
frankfu
rters were manufactured with SL plus 
SD
, 
dipped
 
in 
an 
organic acid solution
, and
 
stored 
for 40 days 
at 10
o
C.
 
 
For
 
the remaining seven full
-
sodium formulations, those containing 
two
 
or 
three
 
organic salts were more 
inhibitory
 
than those
 
containing a
 
single 
organic salt. 
 
In
 
low
-
sodium 
formulations PI
-
2
,
 
PI
-
3
,
 
and 
LI
-
4
, 
 
Listeria
 
populations increased 
less than
 
2 log
 
CFU/g
 
during 9
0
 
days at 4
o
C and 30
 
days at 
7
o
C days
, 
with 
virtually
 
no inhibition seen 
for 
3
8
 
 
any of the
 
formulations at 10
o
C
 
(
Fig
.
 
2
.
2
)
.
 
 
In contrast
, Barmpalia et al.
 
(2004) found
 
significant 
Listeria
 
reduction
s
 
in frankfurters containing
 
1.8% SL + 0.125% SD during 40 days of storage at 
10
o
C. 
 
The
se
 
differences
 
might be due to the 
combined use of 
high organic acid
 
concentrations
 
plus
 
smoke 
which was not 
used
 
in our study.
 
 
In low
-
sodium and 
unc
ured products, the 
effect
 
of 
SL
 
and 
SD
 
against 
Listeria
 
growth was reduced
 
(Legan et al., 2004; Mbandi and Shelef, 2002; 
Seman et al., 2002)
.
 
 
Listeria
 
was suppr
essed for 28 days in unsmoked and 
uncured bratwurst
 
and
 
up to 84 days in smoked and 
cured bratw
urst
 
(Glass et al., 2002)
.
  
Legan et al.
 
(2004)
 
found
 
that their predict
ive
 
growth 
model 
for
 
Listeria
 
work
ed better 
for cured 
than uncured and low
-
sodium products. 
 
Uninoculated frankfurters 
had 
initial 
MAB
 
background 
count
s
 
of 1.
25
 
and
 
2.
57
 
CFU/g 
in 
full
-
 
and low
-
sodium formulations, respectively,
 
(
Fig
.
 
2
.
3
 
and 
2
.
4
).
 
 
M
AB populations in low
-
 
and full
-
sodium uninoculated 
CTR
 
reached 

 
log 
7.0 CFU/g after 30 and 45 days
 
at 4
 
o
C
, 
respectively, which were 1.7 to 2.7 
log 
CFU/g higher than those formulations containing 
Listeria
 
growth inhibitors. 
 
 
At elevated temperatures, 
MAB
 
rapidly grew
 
in the 
uninoculated full
-
sodium
 
frankfurters 
to >
 
7.0
 
log CFU/g 
during 30 days 
at 10
o
C
 
and 45 days at 
7
o
C compared to 15 days 
at 10
o
C
 
for 
the low sodium formulations
. 
 
All nine 
full
-
sodium 
frankfurter formulations containing inhibitors 
yielded 
maximum MAB populations 
of
 
5.
1
 

 
6.
6
 
log CFU/g at 7
o
C 
and 7.0
 
to
 
7.4
 
log CFU/g at 
10
o
C.  
All four low sodium 
frankfurter
 
formulations allowed MAB populations to increase > 7.0 
log CFU/g at 7
o
C
 
and 10
o
C
 
during storage.  
Barmpalia et al. (2004)
 
reported that
 
the
 
t
otal 
microbial count in control frankfurters reached 6.
1 log CFU/cm
2
 
after
 
40 days 
of storage at 
10
o
C
.
 
In contrast,
 
MAB populations 
in our 
non
smoked 
control 
increased
 
to 
6.8 
log CFU/g for 
full
-
sodium 
formulations 
and 7.3
 
log CFU/g for 
low
-
sodium 
formulations 
after
 
15 days at 10
o
C. 
39
 
 
These
 
variations in growth ar
e again
 
expected 
based on
 
the differences 
in formulation, smoking, 
and salt content.
 
In general, 
similar growth trends were seen for
 
MAB 
and 
Listeria
 
on 
frankfurters 
regardless of the formulation, confirming the findings of 
Patel et al.
 
(2009)
.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
40
 
 
 
 
 
 
Figure 
2.1
 
 
Population
 
of 
L.
 
monocytogenes
 
on vacuum
-
pa
ckaged full
-
sodium frankfurters 
for
mulated with powdered or liquid
 
inhibitors
a
 
during 90 days of storage at 4 (A), 7 
(B) and 10
o
C (C).  
 
a
 
Inhibitors in the formulations as in Table 
2.
3
.
 
 
0
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
A: 4
o
C 
 
0
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
B: 7
o
C
 
0
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
storage (days)
 
C: 10
o
C 
 
CTR
PI-1
PI-2
PI-3
PI-4
PI-5
LI-1
LI-2
LI-3
LI-4
41
 
 
 
 
 
 
Figure 2.2  Population
 
of 
L.
 
monocytogenes
 
on vacuum
-
pa
ckaged low
-
sodium frankfurters 
for
mulated with powdered or liquid
 
inhibitors
a
 
during 90 days of storage at 4 (A), 7 
(B) and 10
o
C (C).  
 
a
 
Inhibitors in the formulations as in Table 
2.
3.
 
 
 
 
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
A: 4
o
C 
 
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
B: 7
o
C  
 
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
storage (days)
 
C: 10
o
C  
 
CTR
PI - 2
PI - 3
PI - 5
LI - 4
42
 
 
 
 
 
            
 
                         
 
 
 
Figure 2.3  Population of mesophilic aerobic bacteria
 
on vacuum
-
pa
ckaged full
-
sodium 
frankfurters 
for
mulated with powdered or liquid
 
inhibitors
a
 
during 90 days of storage 
at 4 (A), 7 (B) and 10
o
C (C).  
 
a
 
Inhib
itors in the formulations as in Table 
2.
3.
 
 
0
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
A: 4
o
C
 
0
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
B: 7
o
C 
 
0
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
storage (days)
 
C: 10
o
C 
 
CTR
PI-1
PI-2
PI-3
PI-4
PI-5
LI-1
LI-2
I-L3
LI-4
43
 
 
 
 
 
Figure 2.4  Population of mesophilic aerobic bacteria
 
on vacuum
-
pa
ckaged low
-
sodium 
frankfurters 
for
mulated with powdered or liquid
 
inhibitors
a
 
during 90 days of storage 
at 4 (A), 7 (B) and 10
o
C (C).  
 
a
 
In
hibitors in the formulations as in Table 
2.
3.
 
 
 
 
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
A: 4
o
C 
 
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
B: 7
o
C 
 
2
4
6
8
10
0
15
30
45
60
75
90
log CFU/g
 
storage (days)
 
C: 10
o
C 
 
CTR
PI - 2
PI - 3
PI - 5
LI - 4
44
 
 
2.3
.3
    
Sensory analysis
 
 
Most
 
frankfurter 
formulations were evaluated for consumer acceptance
,
 
with the 
exception of PI
-
2
 
(0.5% SL + 0.5% SA) 
in which
 
the SA concentration
 
exceeded the 
USDA 
allow
able maxim
um limit of 
0.25%
 
(Cox et al., 1989)
. 
 
No
 
significant differences in appearance, 
texture, flavor, and overall consumer acceptability 
were seen for
 
ful
l
-
sodium 
frankfurter
s
 
containing
 
sodium
-
 
or potassium
-
based
 
inhibitors
 
(
P
 
> 0.05) 
(Table 2
.
6
);
 
these findi
ngs agree
 
with th
ose from three previous studies
 
(Barmpalia et al., 2004; Blom et al., 1997; Lu et al., 
2005)
. 
 
Islam et al.
 
(2002)
 
reported significantly lower consumer acceptance scores for 
frankfurters that were dipped in a 25% 
SD
 
solution for 1 min
 
(0.
3% SD pick
-
up). 
 
However, the 
strong initial acetic acid 
odor
 
diminished after 3 days of storage, suggesting that no differences in 
acceptance scores would be expected thereafter. 
 
Using a
 
trained panel
,
 
Stekelenburg and Kant
-
Muermans
 
(2001)
 
found
 
that ham
s containing 0.2% 
SD
 
had significantly lower scores 
for
 
odor 
and taste 
than did hams 
containing lower 
concentrations
 
of 
SD
 
(0.1%), 
SL
 
(3.3%)
, buffered
 
sodium citrate
 
(1%), or 
SD
 
(0.1%)
.
 
 
Lu et al. (2005)
 
did not observe any
 
sour 
or
 
meaty 
off
-
flavor 
for fra
nkfurters 
after 3 min of immersion in a 6% 
SD
 
solution (0.08% SD pick
-
up). 
 
The 
SD
 
and 
PD
 
concentration
s used in our study were 

 
0.156% and 

 
0.2, respectively, which were at or 
below the 
SD
 
pick
-
up 
concentration
 
(0.2 
to
 
0.3%), reported to adversely impact sensory 
attributes. 
 
In
 
low
-
sodium frankfurters, 
formulation PI
-
5
 
(0.2% 
PD
 
+ 0.8% 
PA
) received a 
significantly lower score (
P 
< 0.05)
 
for flavor and overall acceptability
 
than 
did the CTR
, with 
a 
similar texture score
 
(Table 
2
.
7
)
.
 
 
Unlike full
-
sodium frankfurters, the lower 
scores
 
for low
-
sodium frankfurters were expected 
because of
 
weaker masking of acetic acid from 
PD
. 
 
Similar
ly, 
Stekelenburg and Kant
-
Muermans
 
(2001) 
reported 
no adverse sensory 
attribute
 
for 
45
 
 
ham containing
 
0.1% but
 
not
 
0.2% 
SD
. 
However,
 
the potassium salt may have a different flavor. 
A trained sensory panel noted
 
a significant increase in bitterness 
when
 
40 
and
 
50% 
KCl 
was used 
as a 
substitution for NaCl 
in fermented sausage
 
(Glass et al., 2002)
 
and marina
t
ed chicken 
breasts
 
(Lee et al., 2012)
, respectively. 
 
To control 
post
-
thermal 
Listeria
 
growth, meat and poultry manufacture
r
s are increasingly 
incorporating 
o
rganic 
salts 
(e.g., 
SL
 
and 
SD
)
 
into product formulation
s
.
 
 
T
hese salts 
were
 
originally 
developed in liquid form (60% solute) 
because they are
 
high
ly
 
hy
gro
scopic
.
 
 
In the 
present
 
study, 
three
 
full
-
sodium 
frankfurter 
form
ulations 
developed 
for 
Listeria
 
inhib
itors 
and 
containing
 
organic salts as powders with diacetate
 
had properties 
similar 
to those of 
formulations 
containing liquid inhibitors. 
 
Formulations in which organic acids were combined as liquids or 
powders were more effective against
 
Listeria
 
than 
we
re formulations with 
single organic salts, 
particularly at lower temperatures. 
 
Low
-
sodium RTE meat products 
represent
 
a greater risk of listeriosis 
for consumers 
due 
to decreased antilist
erial efficacy of organic salts
 
(Glass et al., 2002; Mbandi and Shel
ef, 2002; 
Seman et al., 2002)
.
 
In
 
this study, 
two
 
low
-
sodium 
frankfurter 
formulations 
containing powdered 
inhibitors 
(PI
-
3 and PI
-
5
) 
had
 
similar
 
or greater 
antilisterial activity 
than did those containing 
liquid
 
inhibitor
s,
 
including 2.5% PL
 
plus 
SD, when
 
stored at 4, 7, 
or 
10
o
C for 90 days. 
 
Strong 
inhibition of
 
Listeria
 
and MAB 
was achieved using
 
PA 
and 
PD
 
regardless of storage 
time
 
and 
temperature. 
 
Compared 
with
 
the low
-
sodium 
CTR
, the powder
-
based
 
formulation 
containing
 
0.2% 
PD
 
(PI
-
5
)
 
was similar in
 
ap
pearance and texture but 
had 
lower 
scores for 
flavor and overall 
accepta
bility. 
 
Given th
ese
 
findings,
 
powdered organic salts 
bacterial inhibitors 
based on 
PA
 
and 
PD
 
should provide an attractive 
alternative to liquid inhibitors 
for both full
-
 
and
 
low
-
sodiu
m 
frankfurters
.
 
46
 
 
Table 
2
.
6 
 
Impact
1
 
of powdered and liquid inhibitors
2
 
on sensory properties of 
full sodium 
 
      
f
rankfurters
.
 
S
odium
-
based
 
CTR
 
P
I
-
1
 
P
I
-
3
 
LI
-
1
 
LI
-
2
 
S
tandard Error
 
frankfurters
3
 
(0%)
4
 
(0.5%)
 
(
0.65%
)
 
(
2.5%
)
 
(
2.5%
)
 
 
 
 
 
 
 
 
 
A
ppearance
 
6.35
a
 
6.50
a
 
6.44
a
 
6.44
a
 
6.35
a
 
0.19
 
T
exture
 
6.52
a
 
6.49
a
 
6.76
a
 
6.49
a
 
6.37
a
 
0.21
 
F
lavor
 
6.51
a
 
6.52
a
 
6.80
a
 
6.38
a
 
6.26
a
 
0.21
 
O
verall
 
6.51
a
 
6.43
a
 
6.74
a
 
6.30
a
 
6.15
a
 
0.2
0
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
P
otassium
-
based
 
CTR
 
PI
-
4
 
P
I
-
5
 
LI
-
3
 
LI
-
4
 
Standard
 
Error
 
F
rankfurters
3
 
   
(
0%
)
4
 
(
0.
7
5%
)
 
(1.0
%
)
 
(
2.5%
)
 
(
2.5%
)
 
 
 
 
 
 
 
 
 
A
ppearance
 
6.23
a
 
6.13
a
 
6.27
a
 
6.38
a
 
6.34
a
 
0.12
 
T
exture
 
6.41
a
 
6.29
a
 
6.30
a
 
6.37
a
 
6.35
a
 
0.13
 
F
lavor
 
6.42
a
 
6.30
a
 
6.26
a
 
6.12
a
 
6.39
a
 
0.14
 
O
verall
 
6.30
a
 
6.12
a
 
6.19
a
 
6.10
a
 
6.33
a
 
0.13
 
 
 
 
 
 
 
 
1
Mean values with 
same letters
 
in the same row wer
e 
not 
significantly different (
P
 

 
0.05).
 
2
Inhibitors as in Table 
2.
3
.
 
3
n
 
= 330 observations. 
 
4
Amount of inhibitor
.
 
 
Table 
2
.
7 
 
Impact
1
 
of powdered and liquid 
inhibitors
2
 
on consumer acceptance scores in 
low 
 
      
sodium frankfurters.
 
 
 
 
 
CTR
 
P
I
-
3
 
P
I
-
5
 
LI
-
4
 
S
tandard Error
 
F
rankfurters
3
 
(0%)
4
 
(0.5%)
 
(
0.65%
)
 
(
2.5%
)
 
 
 
 
 
 
 
 
A
ppearance
 
6.3
3
b
 
6.5
2
a
b
 
6.
60
a
 
6.
56
a
b
 
0.1
1
 
T
exture
 
6.
64
a
 
6.
53
a
 
6.
3
6
a
 
6.
55
a
 
0.
1
1
 
F
lavor
 
6.51
a
 
6.5
0
a
b
 
6.
09
b
 
6.
41
a
b
 
0.
12
 
O
verall
 
6.5
0
a
 
6.4
7
a
 
6.
07
b
 
6.
4
0
a
b
 
0.
1
2
 
 
 
 
 
 
 
1
Mean values with 
same letters in the same row wer
e 
not 
significantly different (
P
 

 
0.05). 
2
Inhibitors as in Table 
2.
3
.
 
3
n
 
= 
206 observations. 
 
4
Amount of inhibitor
.
 
 
 
 
47
 
 
2.4
 
Conclusion
 
Overall findings in the study demonstrate that 
various powder
ed
 
organi
c 
acid 
salts
 
showed ei
ther bacteri
cidal 
or 
bacteriostatic effects on frankfurters 
during storage 
depending on 
types of inhibitor 
combination
s
 
and 
the salt
 
concentration
.
  
In full
-
sodium frankfurters, 
three 
powdered formulations containing diacetate were eq
uivalent or superior to four liquid 
formulations for
 
Listeria 
inhibition, 
especially when potassium 
acetate was combined with 
potassium 
di
acetate
.  
M
ultiple organic salts 
in the formulations 
were more 
effective in 
Listeria 
inhibition
 
than those containing 
a single organic salt. 
 
In low
-
sodium frankfurters, 
the 
formulation with potassium acetate and potassi
um diacetate showed the better
 
inhibition against 
Listeria
 
and MAB growth, regardless of storage day and temperature.  
Given these findings, 
powdered orga
nic salts based on potassium ace
tate and potassium diacetate would
 
be an 
alternative inhibitor to 
current 
liquid inhibitors used in RTE meat products.  Further research
 
is 
needed to improve the sensory properties of the applied products while retaining or 
improving 
the antimicrobial efficacy of the applied organic acid salts.
 
 
 
 
 
 
 
 
 
 
 
48
 
 
CHAPTER 
3
: 
ANTILISTERIAL EFFECTS OF DIFFERENT HOP ACIDS IN 
COMBINATION WITH POTASSIUM ACETATE AND POTASSIUM DIACETATE AT 
7 AND 37
ºC
 
 
 
3.1
 
Introduction
 
 
In the previous study 
(Chapter 2), 
nine orga
nic acid salts from Niacet b.v. (Tiel, The 
Netherlands)
 
were evaluated for their antilisterial activities in frankfurters and 
the results 
indicated 
that the mixture of 80% potassium acetate and 20
% 
potassium diacetate
 
(PAPD)
 
showed th
e best antilisterial activity (Sansawat et al., 2013).   However, the product containing 
PAPD had low sensory score compared to that of the control due t
o its acid flavor.  
Hop extracts 
have been reported to possess antimicrobial activity against 
L. monocy
togenes 
(
Schmalreck and 
Teuber, 1975; Todd et al., 1992;
 
Millis et al., 1994
; 
Larson et al., 1996; 
Bhattacharya et al.,2003
; 
Shen and Sofos, 2008; Shen et al., 2009
).  As a result, it will be ideal if 
the negative impact 
of 
PAPD
 
is decreased by combining w
ith hop acid, especially through product formulation. 
 
Hence, 
t
he purpose of this study 
was
 
to evaluate 
antilisterial effect 
and thermal
 
stability 
of eight
 
hop 
acid 
extracts
 
available from Kalsec
®
 
Inc. 
with
/without
 
PAPD 
in t
rypticase 
s
oy 
b
roth with yeast 
e
xtract (TSBYE)
. 
 
Th
e hypothesis of this research i
s
 
that the combination of hop extracts
 
and 
organic acid salts 
will 
bring synergistic effects in 
L. monocytogenes 
inhibition
.
 
 
3.2
 
 
Material and methods
 
3.2
.1
 
Hop acids and potassium acetate/potassium diacet
ate (PAPD)
 
Eight different hop acid
 
extracts
 
and 
one 
potassium acetate
/
potassium diacetate (
80:20, 
PAPD) 
powder
 
were obtained from Kalsec
®
 
Inc. (Kalamazoo, WI)
 
and 
Niacet b.v. (T
he 
Netherlands)
, respectively (Table 
3.
1). 
 
The
 
average 
concentrations of 
hop 

-

-
acid, acid
-
iso and acid
-
tetra 
in the 
extracts were 67
,
 
96, 78 a
nd 7
6
%, while the concentration
s
 
of hop 
49
 
 
potassium salts were 55, 38, 30, and 10% for K
-
rho, K
-
hexa, K
-
iso, and K
-
tetra, respectively
 
(Table 
3.
1)
. 
For the assessment of antilisterial a
ctivity, 
t
he hop acid extracts were dissolved in 
100% ethanol 
and
 
PAPD 
was dissolved 
in sterile distilled 
water 
and filter
-
sterilized
 

Millex® GS Filter Unit, Carrigiwohill Co., Cork, Ireland)
. 
 
 
Table 
3.
1  
Concentrations of eight hop acid extracts
 
and potassium acetate/potassium diacetate.
 
Materials
 
Concentration (%)
1
 
Alpha acid
 

-
acid) 

 
Humulone
2
 
67.2
 
Beta acid
 

-
acid) 

 
Lupulone
2
 
96.0
 
Aci
d form of isomerized alpha acid
 
(acid
-
iso)
2
 
 
77.5
 
Acid 
form of tetrahydroisoalpha acid
 
(acid
-
tetra)
2
 
75
.8
 
Potassiu
m salt of isomerized alpha acid
 
(K
-
iso)
3
 
 
30.3
 
Potassium
 
salt of hexahydroisoalpha acid
 
(K
-
hexa)
3
 
37.8
 
Potassium 
salt of tetrahydroisoalpha acid
 
(K
-
tetra)
3
 
9.6
 
Potassi
um salt of dihydroisoalpha acid
 
(K
-
rho)
3
 
Potassium acetate/Potassium diace
ate
 
54.6
 
80/20
4
 
1
The concentrations of hop extracts specified by Kalsec
®
 
Inc.
 
2
The remainder (or carrier) is primarily non
-
characterized resinous material including tannins, 
fats, polymers, hop acid by
-
products, hydrophobic substances, and moisture due to
 
the removal 
of the solvent to FDA trace limits.
 
3
The remainder (or carrier) is mostly water due to the extraction in
to
 
aqueous solutions. 
 
4
The mixture of 80% potassium acetate and 20% potassium diacetate prepared by Niacet b.v. 
 
 
 
 
 
50
 
 
3.2.2
 
L. monocytogene
s 
strains and inoculum preparation
 
The 
six
-
strain 
L. monocytogenes
 
cocktail containing ~1 x 10
8
 
CFU
/mL was prepared and 
confirmed t
he 
L. monocytogenes
 
population in the inoculum 
as explained in Chapter 2
. 
 
 
 
3.2
.3
 
Antilisterial activity of eight hop extrac
ts at 37
o
C
 
All of eight hop extract
s were individually dissolved 
in 100% ethanol and added to 
TSBYE
 
to achieve
 
a
 
concentratio
n of 
50 ppm
 
(w/v)
.
 
 
For control, 
the
 
same amount of ethanol 
was 
added
 
without hop acid and PAPD. 
 
A
fter inoculati
ng with the six
-
st
rain 
L. monocytogenes 
cocktail at 3
7
o
C
 
for 24 h, appr
opriate
 
serial dilutions in PBS were plated on TSAYE to 
enumerate 
L. monocytog
enes
. 
 
 
3.2.4
 
Determination of synergistic effect
 
of hop acid/PAPD mixtures on inhibition of 
L. 
monocytogenes 
at
 
37
o
C
 
Hop aci
ds (25 ppm) with/without 0.5% PAPD were separately added to 
TSB
YE 
in test 
tubes 
(16 x 150 mm Pyrex
®
 
Culture Disposable Tube, Corning Incorporated, Corning, NY)
.
  
For 
control, 
the
 
same amount of ethanol was 
added
 
without hop acid and PAPD.
  
All tubes were 
i
noculated with 
Listeria 
for approximately 5.0 

 
6.0 log CFU/mL.
  
T
he 
resulting 
solutions 
as 
well as the 0.5, 1, and 0 (control) %
 
PAPD were 
incubated
  
 
at 3
7
o
C
 
for 24 h.  The 
Listeria 
counts were enumerated by plating 
a
ppropriate dilutions
 
in PBS on TSAYE 
and incubating 
at 
3
7°C for 22 
-
 
24 h.  
The synergistic effect of hop/PAPD combination was determined by 
calculating the combination index (CI)
 
(adapted from Chou and Talalay, 198
3)
, which 
was the 
result of 
sum of log
-
reduction (comparing to initial inocula
tion) from individual treatment 
dividing by log
-
reduction from 
combination treatment
.  The results were 
interpreted as 
synergistic (CI 
<
 
1), additive 
(CI = 1), and antagonistic (CI 
>
 
1). 
 
51
 
 
3.2
.5
 
Antilisterial activity of heated hop extracts
 
Hop acids 
at 
25 
ppm
 
with/without 0.5% PAPD were separately added to 
TSB
YE 
in test 
tubes
.  
During 30 min of heating in a 
85
o
C 
water bath, these tubes were removed at 5 min 
intervals, 
immediately
 
placed in ice slurry for chilling to 37
o
C and inoculated with 
Listeria 
for 
app
roximately 5.0 

 
6.0 log CFU/mL. 
 
T
he 
resulting 
solutions 
as well as the 
1,
 
0.5,
 
and 0 
(control) %
 
PAPD were 
incubated with 
the
 
same amount of ethanol that was used to solubilize 
hop acids
. 
 
For antilisterial activity, a
ppropriate dilutions 
were plated 
on 
TSAYE
 
followed by 22 
-
 
24 h incubation at 3
7
°C.
 
 
3.2
.6
 
Minimal inhibitory concentrations (MIC) of hop extractions in TSBYE
 
Different concentrations of the five hop acids (

-

-
acid, acid
-
tetra, K
-
tetra, and K
-
hexa)
 
ranging from 0 to 25 ppm were added 
to TSBYE. 
 
The
 
uninoculated control and five hop 
extracts inoculated with 
L. monocytogenes 
at 5.0 

 
6.0 log CFU/mL were incubated, after which 
optical density (O.D.) was measured at 600 nm to determine the MIC. 
 
 
3.2
.7
 
Antilisterial activity of hop extract
s and PAPD mixtures at 7
o
C
 
To assess antilisterial activity at 7
o
C for 6 days
, 
TSBYE was prepared to contain 5 ppm 
of 
each of 
the five hop acids (

-

-
acid, acid
-
tetra, K
-
tetra, and K
-
hexa)
 
with/witho
ut 0.5% 
PAPD, in addition to 
1
% PAPD, 
0.5
% PAPD and an inhibitor
-
free control.
 
 
The temperature of 
7
o
C
 
was chosen to represent the condition of temperature abuse during storage of foods including 
m
eats. 
 
After inoculating with 
L
. monocytogenes
 
(3.0 

 
4.0
 
log CFU/mL
),
 
t
he 
samples were 
plated daily for 6 days on TSAYE agar to assess antilisterial activity. 
 
 
52
 
 
3.2
.8
 
Statistical analysis  
 
The microbiological data 
from triplicate experiments were
 
convert
ed 
to log CFU
/mL
.  
An
 
analysis of variance (ANOVA)
 
was performed
 
using the mixed procedure of SAS so
ftware (SAS 
Institute, 2002).  
The 
slope of graph of 
Listeria 
population during storage 
at 7
o
C for 6 days 
for 
each treatment 
(growth rate) 
was calculated
 
to
 
better assess the effect of treatment on 
Listeria 
inhibition.
 
 
Statistically 
significant
 
d
ifferences between 
the 
treatments w
ere
 
dete
rmined using 

 
a
 
= 0.05.
 
 
 
 
 
 
 
 
 
 
 
 
 
53
 
 
3.3
 
Results and discussion
 
3.3
.1
 
 
Inhibitory activity of hop acids agai
nst 
L. monocytogenes 
at 37
o
C
 
When TSBYE containing
 
each of 
eight hop extracts
 
at
 
50 ppm
 
was 
inoculated
 
with the 
six
-
strain 
L. monocytogenes
 
cocktail 
at 5.9 log CFU/mL and 
incubated
 
at 37
o
C
 
for 24 h
, the 
pathogen
 
was below the detection limit
 
of 
1 log CFU/m
L in
 
hop 

-
acid
, with significantly lower 
populations (3.6
 
~
 
3.8
 
log CFU/mL
) seen for 

-
acid, 
acid
-
tetra, K
-
tetra, and K
-
hexa
. 
 
However, 
Listeria
 
grew to 8.6
 
to 
8.9
 
log C
FU/mL
 
in TSBYE containing hop 
acid
-
iso, K
-
iso and K
-
rho, 
which was similar to the cont
rol 
(
P > 
0.05) 
(Fig
. 3
.
1). 
 
King and Ming (2002) also reported 
listericidal effect a
t
 
50 ppm hop 

-
acid
s
 
in trypticase soy broth (TSB)
 
with the population 
decreased
 
by 
4 logs.  
In addition, Larson et al. (1996) and Milles 
and
 
Schendel (1994) reported 
that 
Listeria
 
was 
completely
 
inhibited by 

 
10 ppm hop 

-
acid
s
 
in TSB and brain
-
heart broth, 
respectively, whereas iso
-

-
acids
 
showed little inhibition. 
 
 
Fig
ure
 
3.1



















 

 

-













 

 







 
(The minimum detectability of the methodology was > 10 cells per mi
lliliter). 
 
a,1
 
b
 
b
 
b
 
b
 
c
 
c
 
c
 
c
 
0
1
2
3
4
5
6
7
8
9
10

-
acid
 

-
acid
 
acid-tetra
K-tetra
K-hexa
acid-iso
K-iso
K-rho
TSBYE
log CFU/mL
 
Inhibitors
 
Inoculation at
 
5.87 
log CFU/mL 
 
54
 
 
3.3
.2
 
Synergistic effect of hop acid/PAPD mixtures on inhibition of 
L. monocytogenes 
at
 
37
o
C
 
To evaluate the potential synergistic effects of hop acid/PAPD mixtures, five hop extracts 
(

-
acid
, 

-
acid, 
acid
-
tetra, K
-
tetra, and K
-
hexa
) were selected based on the test results at 50 ppm 
(Fig
.
 
3.
1). 
 
When
 
the
 
mixtures 
of 25 ppm 
hop acid/
0.5% 
PAPD were incubated at 37
o
C with 
Listeria
 
at 5.7 log CFU/mL, the pathogen was no longer detected 
(belo
w detection limit
 
of 1 log 
CFU/mL
) 
in 

-
acid
/PAPD, 

-
acid
/PAPD
, 
and 
acid
-
tetra
/PAPD
 
after 
24 h
, whereas trace levels 
(1.4 

 
2.0 log CFU/mL) of 
Listeria
 
were seen in 
K
-
tetra
/PAPD
 
and K
-
hexa
/PAPD (Fig
.
 
3.
2). 
 
In 
case of single addition, a significant listeri
cidal effect was observed in 

-
acid
 
by reducing the 
Listeria
 
populations 
from 5.7 to 1.8 log CFU/mL, with reduction to 3.6 

 
4.2 log CFU/mL in 

-
acid, 
acid
-
tetra, K
-
tetra, K
-
hexa
, 0.5% PAPD and 1% PAPD (Fig
.
 
3.
2).
 
 
No inhibitor 
control 
allowed the 
Listeria
 
to grow to 
9.2 log
 
CFU/m
L.  The synergistic effect in 
Listeria 
inhibition was 
found when hop acid was used in combination with PAPD except 

-
acid
/PAPD
 
(Table
 
3
.2)
 
(See 
CI calculation in appendix B)
.
  

-
acid
/PAPD
 
combination was 
observed potentially 
due to 
the 
limit detection of the method.
 
These results support previous findings of complete 
Listeria
 
inhibition in TSBYE at 4
o
C 
when 3 ppm hop 

-
acid
 
w
as combined with 1.0% potassium
 
lactate
, 0.25% 
sodium diacetate, or 
0.1% acetic ac
id (Shen and Sofos, 2008). 
 
Seman et al. (2004) also reported that 
L
isteria
 
populations 
decreased
 
from 4.3 log CFU/package to undetectable when hot dogs were dipped in 
an 
antibacterial
 
solution containing 20,000 ppm hop 

-
acid
, 0.3
 
M potassium lactate, and
 
0.3% 
lactic acid in polypropylene glycol.  
 
55
 
 
 
Fig
ure
 
3.2
 
 












 



 

-










 


 





 
 
(The minimum detectability of the methodology was > 10 cells per milliliter). 
 
             
  
 


 
 
 
 
 
 
 
 
 
 
 
 
a
 
bc
 
b
 
bc
 
c
 
a, 1
 
a, 1
 
a, 1
 
a
 
a
 
bc
 
bc
 
d
 
0
1
2
3
4
5
6
7
8
9
10
log CFU/mL
 
Inhibitors
 
Inoculation at
 
5.74
 
log CFU/mL 
 
 
56
 
 
Table 3
.2  
Interpretation
1
 
effects
 
of
 
















 
 
Treatment
 
L
og
-
reduction from 
hop acid or PAPD 
alone
 
Log
-
reduction from 
hop/PAPD
 
Sum of log
-
reduct
ion 
from hop acid alone 
and 
PAPD
 
alone
 
C
I
3
 
Interpretation
 

 
3.94
 
4.74
 
5.53
 
1.17
 
Antagonistic
 

 
2.00
 
4.74
 
3.59
 
0.76
 
Synergistic
 
Acid
-
tetra
 
2.14
 
4.74
 
3.73
 
0.79
 
Synergistic
 
K
-
tetra
 
1.98
 
4.37
 
3.57
 
0.82
 
Synergistic
 
K
-
hexa
 
1.65
 
3.70
 
3.24
 
0.86
 
Synergistic
 
PAPD
 
1.59
 
-
 
-
 
-
 
-
 
1
Data was from Fig.
 
3.2.
 



 



 
 
3.3
.3
 
Thermal stability of hop acid with/without PAPD at 85
o
C
 
Thermal stability of hop acid
s
 
is critically important if the hop inhibitor is 
formulated
 
to
 
meat batters 
to
 
pre
vent 
Listeria
 
growth after cooking. 
 
To evaluate
 
the 
heat stability
, 

-
acid
, 

-
acid, 
acid
-
tetra, K
-
tetra, and K
-
hexa
 
were dissolved in 100% ethanol, diluted to 25 ppm 
with/without 0.5% PAPD
, 
and 
submerged to a water bath 
at 
85
o
C 
for up to
 
30
 
min
. 
 
 
Initially and at 5 min
 
interval
, tube
s were removed,
 
immediately
 
c
ool
ed 
to 3
7
o
C
, and 
inocula
ted with 
Listeria
 
at
 
5.0
 
log CFU
/mL
. 
 
When the 
resulting
 
s
amples
 
were incubated 
overnight at 37
o
C,
 
Listeria 
populations decreased
 
to
 
2.0 and 2.
9
 
log CFU/mL in 

-
acid
 
when 
heated for 10 and 30 min at 85
o
C, respectively. 
 
Listeria
 
populations
 
in the remaining hop acids 
decreased to 3.5 

 
3.8 log CFU/mL, regardless of the heating time (Table 
3.
2). 
 
When PAPD and 
hop acid were mixed, 
L
. monocytogenes 
was 
not 
detected
 
regardless
 
of the heating time, except 
K
-
tetra/PAPD and K
-
hexa/PAPD, which redu
ced
 
the 
pathogen
 
to 1.2 

 
2.1 log CFU/mL (Table 
57
 
 
3.
2). 
 
Number of 
Listeria
 
inoculum was decreased to 3.9 

 
4.0 and 3.5 

 
3.6 log CFU/m
L
 
in 0.5 
and 1% PAPD, respectively, regardless of the heating time, 
where
as the 
control
 
allow
ed
 
Listeria 
to grow
 
to 9.4 log
 
CFU/mL
 
after
 
incubation
 
at 37
o
C
 
for 24 h
.     
 
 
 
 
 
 
 
 
 
58
 
 
Table 3.
3
 
Popula
tion of 
L. monocytogenes
1
 
in TSBYE
2
 
containing 25 ppm hop acid extracts with/without 0.5% PAPD
3
 
after heating 
at 85
o
C.
 
Treatment
 
Populations of 
L. monocytogenes
4
 
(log CFU/mL) in TSBYE
 
cooked various time (min)
 
Min 0
 
Min 5
 
Min 10
 
Min 15
 
Min 20
 
Min 25
 
Min 30
 

 
1.92 

 
0.24
 
a
 
1.84 

 
0.16
 
a
 
1.96 

 
0.36
 
ab
 
2.19 

 
0.28
 
abc
 
2.76 

 
0.51
 
bc
 
3.01 

 
0.03
 
c
 
2.87 

 
0.20
 
c
 

 
3.54 

 
0.38
 
3.50 

 
0.32
 
3.59 

 
0.24
 
3.67 

 
0.19
 
3.81 

 
0.18
 
3.83 

 
0.11
 
3.75 

 
0.17
 
Acid
-
tetra
 
3.46 

 
0.27
 
3.50 

 
0.17
 
3.57 

 
0.11
 
3.51 

 
0.17
 
3.46 

 
0.13
 
3.63 

 
0.13
 
3.58 

 
0.13
 
K
-
tetra
 
3.64 

 
0.07
 
3.65 

 
0.08
 
3.65 

 
0.08
 
3.63 

  
0.07
 
3.63 

 
0.09
 
3.55 

 
0.09
 
3.61 

 
0.04
 
K
-
hexa
 
3.58 

 
0.06
 
3.56 

 
0.09
 
3.56 

 
0.01
 
3.53 

 
0.02
 
3.51 

 
0.05
 
3.51 

 
0.14
 
3.50 

 
0.13
 

 
ND
5
 
ND
 
ND
 
ND
 
N
D
 
ND
 
ND
 

 
ND
 
ND
 
ND
 
ND
 
ND
 
ND
 
ND
 
Acid tetra + PAPD
 
ND
 
ND
 
ND
 
ND
 
ND
 
ND
 
ND
 
K
-
tetra + PAPD
 
1.20 ± 0.07
 
1.38 ± 0.35
 
1.53 ± 0.10
 
1.37 ± 0.14
 
1.39 ± 0.16
 
1.48 ± 0.03
 
1.66 ± 0.04
 
K
-
hexa + PAPD
 
1.35 

 
0.33
 
1.69 

 
0.27
 
1.76 

 
0.19
 
1.70 

 
0.43
 
1.99 

 
0.
36
 
2.06 

 
0.66
 
2.08 

 
0.68
 
0.5%PAPD
 
3.88 

 
0.20
 
3.94 

 
0.16
 
3.94 

 
0.10
 
3.94 

 
0.08
 
3.94 

 
0.18
 
3.99 

 
0.18
 
3.97 

 
0.16
 
1%PAPD
 
3.48 

 
0.06
 
3.43 

 
0.10
 
3.43 

 
0.10
 
3.48 

 
0.14
 
3.54 

 
0.12
 
3.49 

 
0.12
 
3.61 

 
0.13
 
TSBYE
 
9.41 

 
0.14
 
 
 
 
 
 
 
1
Listeria 
inocula
ted with 5.01 
+
 
0.53 log CFU/mL
.
  
2
T
SBYE, Trypticase soy broth with yeast extract.
 
3
PAPD, Potassium acetate and potassium diacetate.
 
4
Means 

 
standard deviation of 
n
 
= 6 observations
 
for each reading
.
  
5
ND, Not detected
.
 
a
-
c
Mean values with same letters in
 
the same row were not significantly different (
P
 

 
0.05).
 
 
59
 
 
3.3
.4
 
Minimal inhibitory concentrations of hop acids against 
Listeria 
growth at 37
o
C
 
When 
Listeria
 
inoculum (
5.1
 
log CFU
/mL
)
 
was incubated
 
at 37
o
C
 
for 24 h
, optimal 
density (O.D.) 
at 600 nm
 
incre
ased to 0.8.
 
 
Using the method, m
inimal inhibitory concentrations 
(MIC) against 
Listeria
 
were determined with no increase of O.D. 
The MIC was 6.3 ppm for 

-
acid
, 

-
acid, acid
-
tetra
, 
with
 
12.5 ppm 
seen 
for 
K
-
tetra and K
-
hexa (Table 
3.
4
)
. 
 
These results are 
consistent with a previously reported MIC of 
~
 
6 ppm for hop
 

-
acid
 
(Mill
i
s and Schendel, 1994; 
Barney et al., 1995).  
 
 
Table 3.4
  
Minimal inhibitory con
centrations (ppm) of hop acid extracts on 
L. 
monocytogenes
1
 
growth.
 
Treatment
 
Growth of 
L.
 
monocytogenes
 
(O.D.)
2
 
Hop acid concentration (ppm)
 
25
 
12.5
 
6.3
 
3.1
 
1.6
 
0.8
 
0
 

-
acid
 
0.000
 
0.000
 
0.000
 
0.005
 
0.432
 
0.530
 
0.799
 

-
acid
 
0.000
 
0.000
 
0.000
 
0.004
 
0.
008
 
0.392
 
0.799
 
acid
-
tetra
 
0.000
 
0.000
 
0.000
 
0.004
 
0.547
 
0.716
 
0.799
 
K
-
tetra
 
0.000
 
0.000
 
0.010
 
0.546
 
0.712
 
0.748
 
0.799
 
K
-
hexa
 
0.000
 
0.000
 
0.336
 
0.704
 
0.755
 
0.761
 
0.799
 
1
 
Initial inoculation of 
L
.
 
monocytogenes
 
was 
5.11 

 
0.28 log CFU/mL.
 
2 
O.D. Optical
 
density at 600 nm: the growth of 
Listeria
 
in TSBYE after incubation at 37
o
C for 
 
24 h.  
 
 
 
 
 
 
60
 
 
3.3
.5
 
E
ffect of hop acid/PAPD mixtures on inhibition of 
L. monocytogenes 
at
 
7
o
C
 
 
In 
study
 
3.
3.
2
, antilisterial activities of hop acids at 25 ppm, PAPD and their 
c
ombinations were evaluated in TSBYE at 37
o
C. 
 
To meet the USDA allowance for hop acid 
(USDA
-
FSIS, 2013) and to simulate the condition of temperature abuse during food storage, the 
antilisterial activity of hop acids at 5 ppm were evaluated with/without 0.5
% PAPD during six 
days of storage at 7
o
C. 
 
In addition to the hop acids
 
and hop acid/PAPD mixtures, 1% PAPD, 
0.5
% PAPD and inhibitor
-
free control were included. 
 
A
ll mixtures of 0.5% PAPD/5 ppm hop 
acid and single addition of 1% PAPD, 0.5% PAPD, and
 
5 ppm 

-
acid showed listeristatic effect 
with similar slope of 
Listeria 
growth curve 
(
P 

 
0.05)
 
(Fig. 3.3)
.
 
 
However, the 
remaining 
hop 
acids and control allowed the pathogen to grow 
from 3.8 log CFU/mL to 5.6 

 
7.3
 
log CFU/mL
 
at the end of storage (Fig. 3.3 and
 
Table A.7)
. 
 
     
 
 
 
 
 
 
 
 
61
 
 
 
 
Fig
ure
 
3.3
 
 
Population
 
of 
L. monocytogenes
 
 
in TSBYE with or without different hop acid 
extracts at 5 ppm
, 
0.5 or 1% PAPD, 


 
during 6 days of storage at 7
o
C.
 
 

-








 


 
 
The comparison of antilisterial effects between hop acids at 25 ppm/37
o
C and hop acids 
at 
5 ppm/7
o
C, with/without 0.5% PAPD, led to two interesting observations. 
 
Firstly, 

-
acid was 

-
acid at 37
o
C while the opposite was true at 7
o
C. The different activity at two 
different temperatures can be explained by the shorter shelf
-

-
acid at elevated 
temperatures, presumably due to oxidation.
 
 
Using HP
LC and well diffusion assay, Seman et al. 
(2004) demonstrated that pho
to
-
oxidation decreased the anti

-
acid 
compared to that with antioxidants. 
 
Using
 
chelating or antioxidant agents, King and Ming 
(2002) also reported stronger an

-
acid compared to 
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
log CFU/mL
 
Storage time (days)
 
K-tetra
K-hexa
acid-tetra

-
acid
 

-
acid
 
0.5% PAPD
1% PAPD
TSBYE
K-tetra/PAPD
K-hexa/PAPD
Acid-tetra/PAPD

-
acid/PAPD
 

-
acid/PAPD
 
b
 
a
 
62
 
 

-
acid alone at 30
o
C for 48 h in trypticase soy broth. 
 
Regardless of incubation temperature, 
however, the mixtures of hop acid and PAPD showed consistent listericidal effect. 
 
Secondly,
 
the combination of 0.5% PAPD /25 ppm hop acid showed 
stronger listericid
al 
activity at 37
o
C compared to single application of hop ac

-
acid (Fig.
 
3.
2). 
 
Interestingly, no synergistic effect was found using the combination of 0.5% PAPD/5
 
ppm hop 
acid at 7
o
C (Fig.
 
3.
4). 
 
These results could be explained if the amount 
(5 ppm) of hop acids was 
not 
sufficient to generate inhibition or synergistic effects at 7
o
C. 
 
Larson et al. (1996) reported 
that 
Listeria
 
growth was similar in the skim milk 
control and the milk containing 1 and 10 ppm 

-
acid during 30 days of storage at 4
o
C. 
 
A listericidal effect was seen only when the 

-
acid was increased to 100 and 1,000 ppm.  
 
 
 
 
63
 
 
3.4
 
Conclusion
 
In evaluation of 
Listeria
 
inhibition, fi

-

-
acid, acid
-
tetra, K
-
tetra, and K
-
hexa) 
out of 8 hop extracts were more antilisterial than the remaining three (
acid
-
iso, K
-
iso,
 
and 
K
-
rho
)
. 
 
The minimal inhibitory concentration of hop acids 

-

-
acid, and 
acid
-
tetra, w
ith < 12.5 ppm seen for K
-
tetra, and K
-
hexa. 
 

-
acid 
was most inhibitory against
 
L. monocytogenes
 
at 37
o
C
 
regardless of heating at 85
o

-
acid demonstrated the best antilisterial activity at 7
o
C. PAPD alone inhibited 
L
isteria
 
more 
effectively at 7 than at 37
o
C, probably due to the high growth rate of 
Listeria
 
at 37
o
C. 
 
Regardless of incubation temperature and addition amount, the mixtures of hop acid/PAPD 
resulted in more robust inhibition than did any single addition. 

-
acid, 

-
acid, acid
-
tetra, K
-
tetra, and K
-
hexa) showed listericidal activity at 37
o
C, whereas the hop 
acids at 5 ppm allowed
 
Listeria
 
to grow at 7
o

-
acid, potentially due to insufficient 
amount. 
 
Based on this findings
, the single addition of hop acids at 5 ppm appears to
 
be not 

-
acid, to inhibit 
Listeria 
at 7
o
C while the mixture of hop/PAPD provide a 
better inhibition.   
 
 
 
 
 
 
 
 
 
 
64
 
 
CHAPTER 
4
: 
INHIBITION OF 
LISTERIA MONOCYTOGENES
 
IN DELI
-
STYLE 
TURKEY 
AND MILK USING HOP ACID EXTRACTS WITH
 
OR WITHOUT
 
POTASSIUM ACETA
TE AND POTASSIUM DIACETATE
 
 
4
.1
 
Introduction
 
Hop
 
acids have long been known for antimicrobial activity against Gram positive bacteria 
(
Sakamoto
 
and Konings, 2003
).  
Previously
, the 
antimicrobi
al 
activity 
of hop acids 
has 
predominantly 
been 
assessed in liquid media or 
on the 
food
s
 
after surface application 
rather than 
after
 
product formulaiton
.  
More s
pecifically, no research
 
has been conducte
d to evaluate 
antimicrobial activity of h
op or hop/or
ganic acid combinati
on
s in 
processed meat
 
formulation
.
  
A
lthough RTE meat pro
ducts are fully cooked, the additional
 
contamination may occur during
 
post
-
thermal
 
h
andling and storage.  Therefore
, 
this study was designed to assess 
antilisterial 
activity of
 
h
o
p or hop/organic acid combinati
ons
 
in the practical situation
 
at 
manufacture plant
s
 
and at deli store
s
 
or at home
s
 
(F
i
g.
 
4.1).
  
After manufacture of product, the 
time 
required 
for deli 
meat distribution to retail
 
s
tore
 
is
 
about
 
10 to 30 days
 
(US
DA
-
FSIS
, 20
03
c
)
.  After delivered to
 
retail stores, the deli meat is usually
 
display
e
d
 
for 5 to 30 days d
epending on 
sale (Personal 
interview)
.  After purchase
, more
 
than 75% of consumers 
are likely consuming
 
deli meat
s
 
within 
1 to 10 days (AMI, 2000).  
  
 
 
 
 
 
 
 
 
65
 
 
 
 
  
  
 
 
 
 
 
 
 
 
Figure 4.1
  
Expected times for production, distribution, and consumption of d
elicatessen meat
s. 
 
 
In our previous study (chapter 3), the combination of hop
 
acid and PAPD demonstrated 
additive 
or synergistic 
effects on 
Listeria 
inhibiti
on in liquid media.  
Therefore, the objective of 
this study was to evaluate the antilisterial activity of 
hop extracts with/without PAPD 
in 
deli
-
style turkey meat.  
The hypothesis of this research is
 
that that 
the 
mixture of hop 
acid 
and 
PAPD 
effectively i
nhibits 
Listeria
 
in deli
-
style
 
turkey meat
 
not only upon product
ion but also during the 
distribution and storage. 
 
 
4.2
 
Material and methods
 
4.2
.1
 
Deli
-
style turkey 
preparation with
/without 
Listeria
 
inhibitors  
 
Turkey breasts and ingredients 
required 
for
 
deli
-
style turkey 
were obtained locally.  
Hop 
aci
d 
extracts and PAPD were obtained 
from 
Kalsec Inc.
 
(
Kalamazoo, MI) 
and 
Nia
cet b.v. (Tiel, 
The Netherlands), respectively, while OptiForm was purchased from PURAC America, Inc 
(Lincolnshire, IL)
.
 
 
D
eli turkey
 
meat
 
was traditionally 
manufactured
 
a
t the Michig
an State 
University (MSU) Meat L
aboratory (East Lans
ing, MI), 
using the 
following 
formuation
: turkey 
breast
 
(71.84%), water (20.44
-
21.94%), salt (1.68
%), phosphate (0.36%), starch (2.5
0
%), suga
r 
Manufa
cturer
 
Deli store/Retail
 
Home
 




-



 




 




 
-
 
39% used within 1
-
3 days
 
-
 
36% used within 4
-
7 days
 
-
 
3% used within 8
-
10 days
 
.
 
.
 
.
 
-
 
0% used within 
>
 
61 days
 
66
 
 
(1.44%), so
dium nitrite (0.18
%), erythorbate
 
(0.0578
%)
 
and 
inhibitor
 
(0
-
2.5%)
.  
Seven 
Listeria
 
inhibitors 
were 
prepared 
as previously 
described 
and 
te
sted 
as follow:  (1
)
 
inhibitor
-
free 
control 
(CTR); (2
) 2.5 % OptiForm
®
 
solution of 56% potassium lactate, 4% sodium d
iace
tate, and 40
%
 
water (PLSD); (3
) 0.5% 
powdered mixture of potassium acetate
 
(80%)
 
and 
potassium d
iacetate 
(20%) (PAPD); (4

-
acid
,
 

-
acid)
; (5
) 5 ppm
 

-
acid
 

-
acid
/PAPD
)
; (6

-
acid

-
acid
)
; and 
(7

-

-
acid
/PAPD
)
. 
 
  
 
Tu
rkey breast 
meat was
 
ground and 
mixed
 
with 
the 
required
 
ingredients 
in a bowl 
chopper (model K64
-
Va, Maschinenfabrik Seydelmann KG, Aalen, Germany) 
under vacuum 
for 
8 min.  T
he 
meat batter was
 
stuffed into 
fibrous 
casing
s
 
(
90 mm
; Devro
-
Teepak Inc.,
 
Danville, 
IL
)
 
and cooked to an internal temp
erature of 74
o
C in a smoke
-
free smokehouse. 
 
After cooking
 
and
 
showering
, the deli turkey chubs
 
were
 
stored overnight at 2
o
C
 
and sliced for 
physicochemical and microbial analyses.  
  
 
 
4.2
.2
 
Physicochemical analysis of deli turkey meat 
 
Deli turkey meat wa
s
 
analyzed for pH, water activity (a
w
), moisture, fat, and cooking 
yield.  For pH, a 5
-
g sample was homogenized in 25 mL of deionized water and then pH was 
measured with a meter (Accumet AR15, Fisher Scientific, Pittsburgh, PA) equipped with a pH 
electrode
 
(model 13
-
620
-
631, Fisher Scientific, Houston, TX).  Water activity (a
w
) was 
determined with an AquaLab meter (Decagon Devices, Pullman, WA).  
Moisture and fat 
contents were determined 
with drying oven 
(model Yamato DX 400, Yamato Scientific. Ltd.,
 
Tokyo,
 
Japan), and fat extractor (Soxtec System HT6, Tecator AB, 
Höganäs
, Sweden), 
respectively,
 
according to the AOAC International official methods 950.46B and 991.36, 
67
 
 
respectively (AOAC, 2005).  The cooking yield of deli turkey was determined based on the 
wei
ght difference before and after cooking. 
 
 
4.2
.3
 
Listeria
 
monocytogenes
 
strains and 
inoculum preparation
 
 
 
The cocktail of six 
L. monocytogenes 
strains was prepared to contain ~1 x 10
8
 
CFU/mL 
as explained in Chapter 3.  
The cocktail was then serially dilut
ed in sterile phosphate
-
buffered 
saline (PBS; pH 7.4) to a level of approximately 
10
5
 
CFU/mL of 
L
.
 
monocytogenes
 
for deli meat 
inoculation.  The 
L. monocyt
o
genes
 
population 
in 
the 
inoculum was enumerated 
by
 
plating 
appropriate dilution
s
 
on Trypticase soy a
gar 
with yeast extract 
(TSA
YE
) 
followed by 
24 h 
incubation 
at 37
°
C.  
 
 
4.2
.4
 
Deli turkey meat i
noculation 
 
 
Listeria
 
inoculation of the
 
deli turkey meat was conducted in two different w
ays to 
simulate contamination in the 
plant 
during manufacture and 
at 
re
tail 
deli
s
 
or 
in the 
home.  
For
 
contamination during manufacture
, the deli meats were sliced
 
(approximately 1.5 mm thick
 
and 
25 
+
 
1 g weight) with 
a mechanical delicatessen slicer (
model 410, Hobart, Troy, OH
)
 
and spot 
inoculated at several location
s on on
e side with 0.1 mL to obtain 2
-
3 log CFU/g.  
The slices were 
then placed in
 
a biological safety cabinet for 20 min to 
allow the inoculum absorb
.  Four slices 
were placed 
in 
each 
bag
 
(18 by 30 cm; VacMaster, Kansas city, MO),
 
vacuum packaged
 
(Multivac, Sepp
 
Haggenmueller GmbH & Co. KG., Wolfertschwenden, Germany
), and store
d
 
at 
4 and 7
o
C.
  
For contamination at 
retail delis
 
or 
at 
home, 
the 
cooked 
chub
s were 
first stored
 
for 30 
and 60 days 
4
o
C
, and
 
then 
slice
d and 
inoculated as above
.  
Four slices were placed 
on 
a 
piece of 
68
 
 
delicatessen paper (20 
x 
27 cm; Brown Paper Goods, Waukegan, IL),
 
aseptically transferred to 
a 
zip lock
 
delicatessen bag (20 
x
 
25 cm; Elkay Plastics)
, and stored 
at 
4 and 7
o
C
 
for 10 days
.
  
 
 
4.2
.5
 
Microbiological analysis  
 
Turkey slices 
from
 
the day of ma
nufacture were assessed for 
initial 
populations of 
L. 
monocytogenes
,
 
and then tested at 
15 da
y intervals 
for up to 60 days.  S
lices prepared after 30 
and 60 day
s of
 
storage were analy
zed for the pathogen
 
ini
ti
ally and every 2 days 
up to 10 da
ys.  
For each treatment, duplicate 25
-
g samples were diluted 1:10 in PBS and homogenized in a 
stomacher (NEUTEC Group, Farmingdale, NY) for 2 min.  Appropriate serial dilutions in PBS 
were plated on modified Oxford agar (MOX) (Difco, BD) to enumerate 
L. mo
nocytogenes
 
after 
incubation 
for 
48 h at 
37
o
C.
  
 
 
4.2
.6
 
Antilisterial activity of hop extracts and PAPD mixtures 
in milk 
at 7
o
C
 
 
 
T
he antilisterial activity of hop acids was assessed in skim 
milk 
and 2% milk.  Both ski
m 
milk 
and 2% milk (Meijer, Grand Rapi
d
s, MI
) were purchased lo
cally, to which 
hop 

-

-
acid, acid
-
tetra, K
-
tetra, and K
-
hexa
 
were 
added
 
separately 
with/without 0.5% PAPD
 
as well as 
0% 
(
control
)
 
and 
0.5% PA
PD.  The
se
 
samples were inoculated with 
the six
-
strain 
L.
 
monocytogenes 
cocktail so as to contain
 
approximately 3.0 

 
4
.0 log CF
U/mL
 
and incubated at 
7
o
C for 6 days
.  The 
L.
 
monocytogenes 
populations were
 
then
 
enumerated daily by plating an 
a
ppropriate serial dilution in PBS on MOX and incubating at 
37
o
C
 
for 48 h
.
 
 
 
 
69
 
 
4.2
.7
 
Statistical analysis
  
 
All
 
experiment
s were conducted in tr
i
plicates
.  The microb
iological data were converted 
to log CFU/g (for deli meat) or log CFU/mL (for milk) and analyzed using the mixed procedure 
of 
SAS software (SAS 
Institute, 2002).  To better assess the effect of treatment on 
Listeria 
inhibition, the sl
ope 
of graph of 
Listeria 
population 
during storage 
for each treatment (growth 
rate) 
was calculated.  
Mean d
ifference
s
 
of 
L. monocytogenes 
population or slope 
of the graph 
between treatment
s
 
were
 

a
 
= 0.05 
level.
  
For the com
parison of 
skim milk
 
and 2% milk,
 
the student
 
t 
test was used for
 
a
 
paired
-
wise comparison for
 
Listeria 
growth
.  
 
 
4.3
 
Results and discussion
 
4.3
.1
 
Physicochemical properties of deli turkey meat
 
 
Deli
-
style turkey
s
 
containing 7 inhibitors including 
the 
con
trol were assessed for pH, 
a
w
, 
moisture, fat, and cooking yield.
  
No significan
t differences were found among the seven 
inhibitor treatments regardless of 
the evalu
a
tion 
parameter 
(
P 
>
 
0.05, Table 4.1).  The pH and 
a
w
 
ranged from 6.26 to 6.30 and fr
om 0.96
6 to 0.972, respectively, which were similar to the
 
values 
found previously in 
deli tu
rkey meats prepared with/witho
ut common organic acid mi
xture 
(
Zhang et al.
, 
2012)
.
 
 
Shen et al. (2009) also reported that 
dipping
 
frankfurter
s
 
in 0.03 

 
0.10% 
hop 

-
acid
 
solutions did not significantly change the pH and 
a
w
 
values compared to un
dip
ped
 
control
.  I
n general, our results indicate
 
that 
the six antimicrobial agents 
did not affect 
(
P 
>
 
0.05)
 
physicochemical properties 
of deli
-
style turkey
.
  
  
 
 
 
70
 
 
Table 4.1
  
Physicochemical prope
rties of deli
-
style turkey mea
t 
 
Inhibitor
 
\
Parameter
 
1
 
pH
 
a
w
 
Moisture 
 
(%)
 
Fat 
 
(%)
 
Cooking yield 
(%)
 
CTR
 
6.30
 
+
 
0.04
 
0.968
 
+
 
0.007
 
72.96
 
+
 
0.91
 
0.97
 
+
 
0.37
 
85.93
 
+
 
2.42
 
PLSD
 
6.30
 
+
 
0.04
 
0.966
 
+
 
0.005
 
71.34
 
+
 
1.23
 
1.01
 
+
 
0.41
 
87.0
2
 
+
 
3.48
 
PAPD
 
6.27
 
+
 
0.05
 
0.970
 
+
 
0.003
 
71.69
 
+
 
1.61
 
0.97
 
+
 
0.47
 
85.04
 
+
 
3.33
 

-
acid
 
6.30
 
+
 
0.06
 
0.971
 
+
 
0.005
 
71.71
 
+
 
0.45
 
0.97
 
+
 
0.41
 
83.90
 
+
 
0.92
 

-
acid/PAPD
 
6.29
 
+
 
0.04
 
0.970
 
+
 
0.001
 
72.31
 
+
 
1.66
 
0.99
 
+
 
0.44
 
84.78
 
+
 
0.44
 

-
acid
 
6.30
 
+
 
0.06
 
0.972
 
+
 
0.004
 
72.94
 
+
 
1.33
 
0.99
 
+
 
0.38
 
86.47
 
+
 
0.03
 

-
acid/PAPD
 
6.26
 
+
 
0.07
 
0
.972
 
+
 
0.0
05
 
72.82
 
+
 
1.09
 
1.01
 
+
 
0.36
 
85.63
 
+
 
0.91
 
1
Number of observations for
 
eac
h parameter per inhibitor, 
n = 9
 
except for
 
cooking yield (
n = 2
)
.
 
 
CTR: I
nhibitor
-
free
 
control
.
 
PLSD: 2.5% of potassium lactate (56%)/sodium diacetate (4%)/water (40%)
.
 
PAPD: 0.5% of
 
potassium acetate (80%)/potassium diacetate (20%)
.
 

-
acid

-
acid
.
 

-
acid

-
acid
/0.5% PAPD
.
 

-
acid
: 5 ppm of hop
 

-
acid
.
 

-
acid

-
acid
/0.5% PAPD
.
 
 
 
 
 
 
 
 
 
 
71
 
 
4.3
.2
 
L. monocytogenes
 
on deli turkey meat
 
and in milk
 
S
pot i
noculation 
resulted in 
L. monocytogenes
 
populations o
f
 
2.28 to
 
2.59
 
log CFU/g 
in 
deli
-
style turkey 
(Fig.
 
4.2
 
and Table A.8
).  
Deli
-
style turkey formulated with 
PLSD
, PAPD,
 

-
acid
/PAPD

-
acid
/PAPD 
showed s
i
milar trend
s
 
for 
Listeria 
growth 





, 
inhibiting
 
the 
growth of 
L. monocytogenes 
for 60 days 
d
uring storage at 
4
o
C
 
(Fig. 4.2).  
However, 
the 
addition 
of 

-
 
or
 

-
acid
 
allowed the pathogen 
populations to increase
 
> 4.5 log CFU/g, which wa
s not 
significantly different from the control (
P
 
< 0.05) (Fig.
 
4.2
 
and Table A.8
).
 
When stored at 
7
o
C
 
for 60 days
, 
again, the trends of 
Listeria 
growth for PLSD, PAPD, 

-
acid

-
acid
/PAPD were similar 







  
B
oth 

-
acid
/PAPD 

-
acid
/PAPD allowed 
Listeria 
to gro
w < 2.0 log CFU/g, whereas 
increase of 
2.2 to
 
3.2 and 5.
6 to
 
5.8 log 
were seen f
or
 
two 
organic acid 
mixtures (PAPD and PLSD) and two
 

-
 

-
acid
), respectively, w
ith 
a 
5.9 log 
increase
 
seen for 
the 
inhibitor
-
free
 
control (Fig.
 
4.2
 
and 
Table A.8
).  
When used individually, 
Bedie et al. (2001) reported that 
6%
 
sodium
 
l
actate 
and 
0.5% 
sodium diacetate
 
were
 
listeristatic or listericidal 
in frankfurters during 
120 days
 
of storage
, 
while half the 
concentration prevented 
gr
o
wth 
of 
L. monocytogenes 
for
 
50 to 70 days
.  When 
used 
organic acid salts, our 
previous research 
show
ed that 
five 
of 
nine organic acid mixtures 
were
 
listericidal 
on frankfurters 
during
 
90 days
 
of storage
 
at 4
o
C, whereas only one 
(PAPD) 
of 
five 
organic acid mixtures 
maintained listericidal activity
 
when th
e storage temperature was 
increas
ed to
 
7
o
C (
Fig. 2.
1
).  Similarly, Blom et
 
al (1997) demonstrated that a
 
mixture of 2.5% 
sodium lactate and 0.25% sodium acetate inhibited 
the 
growth of 
L. monocytogenes
 
in sliced 
cooked ham throughout 5 weeks 
of storage 
at 4
o
C, but only 2 to 3 weeks at 9
o
C.  
 
According to t
he 
USDA
-
FSIS definition for an
 
anti
microbial agent
 
(
USDA
-
FSIS
, 
2003),
 
such an
 
antimicrobial agent is a substance that 
effectively reduces
, 
eliminates
, 
or 
suppress
es
 
72
 
 
microbial
 
growth throughout the shelf life of the products
.  As a result,
 
the agent
 
shoul
d 
allow no 
more than 2 
logs of growth 
during
 

.  
Therefore, our results suggest
 
that the 
combination of 
5 ppm 

-
acid

-
acid
/0.5% PAPD could be used 
under 
USDA
-
FSIS alternatives 1 or 2 to inhibit 
L. monocytogenes 
in 
ready
-
to
-
eat meat products during 
the storage at 
7
o
C.  One of 
the 
interesting re
sults found in this research is
 
that both PLSD and 
PAPD were very effective in inhibiting 
Listeria 
at 4
o
C but not at 7
o
C.  At
 
7
o
C, 
the mixture of 

-
acid
/0.5% PAPD
 
was the most 
effective with the intermediate seen for 

-
acid
/0.5% PAPD
, 

-
acid

-
acid
, 
 
and
 
control (Fig. 
4.2). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
73
 
 
 
 
 
 
 
Figure 4
.2
  
L. 
monocytogenes
 
populations
 
i
n 
vacuum
-
packaged deli
-
style turkey meat with 
va
rious inhibitors during 60 days of storage at 
4 (A)
 
and 
7
o
C
 
(B)
.
 
 

-







 
 






0
1
2
3
4
5
6
7
8
0
7
15
30
45
60
log CFU/g
 
Storage time (days)
 
A: 4
o
C 
 
a
 
b
 
0
1
2
3
4
5
6
7
8
9
0
7
15
30
45
60
log CFU/g
 
Storage time (days)
 
B: 7
o
C 
 
CTR
PLSD
PAPD

-
acid
 

-
acid/PAPD
 

-
acid
 

-
acid/PAPD
 
a
 
b
 
74
 
 
S
urface inoculation 
of deli
-
style turkey, which was
 
sliced after 30 and 60 days of s
torage 
at 
4
o
C
,
 
yielded average 
L. monocytogenes 
populations of 2.18 to 2.45 log CFU/g and 2.66 to
 
2.78 
log CFU/g, respectively 
(Fig.
 
4.3, 
4.4
 
and Table A.9, A.10
).  
During 10 days of storage at 
4
o
C
, 
Listeria
 
populations 
decreased
 
< 0.6
 
log CFU/g using
 
PLSD
, PAPD, 

-
acid

-
acid
/PAPD, whereas the pathogen
 
 
increased
 
by 0.5 to
 

-
acid
 

-
acid
 
although no si
gnificant difference 
(
P 

 
0.05)
 
was seen
,
 
regardless of treatment
 
(Fig. 4.3
 
and 
Table A.9
).  During 
storage at 7
o
C
,
 
PLSD, 
P
APD
,
 

-
acid
/PAPD, 

-
acid
/PAPD
 
were
 
listeristatic 
with similar trend of 
Listeria 
growth (
P 

 
0.05) 
while 
both CTR 

-
acid
 
allow
ed
 
Listeria 
to gro
w
 
by 1.2 to
 
2.0
 
log CFU/g
,
 
resulting in significantly higher populations 
than the 
other
 
treatments 
(Fig.
 
4.3, 
4.4
, and Table A.9, A.10
). 
 
 
 
 
 
 
 
 
 
 
 
 
 
75
 
 
 
 
 
 
Figure 4
.
3
  
L. 
monocytogenes
 
populations
 
during 1
0 days of storage at 
4 (A)
 
and 
7
o
C
 
(B) 
i
n 
aerobic
-
packaged deli
-
style turkey meat with various inhibitors and 
sliced after 30 
days of storage.
 
 

-








 
 






0
1
2
3
4
5
0
2
4
6
8
10
log CFU/g
 
Storage time (days)
 
(A): 4
o
C
 
a
 
b
 
0
1
2
3
4
5
0
2
4
6
8
10
log CFU/g
 
Storage time (days)
 
(B): 7
o
C
 
CTR
PLSD
PAPD

-
acid
 

-
acid/PAPD
 

-
acid
 

-
acid/PAPD
 
a
 
b
 
76
 
 
 
 
 
 
 
 
 
Figure 4
.4
  
L. 
monocytogenes
 
populations
 
during 1
0 days of storage at 
4 (A)
 
and 
7
o
C
 
(B) 
i
n 
aerobic
-
packaged deli
-
style turkey meat with various inhibitors
 
and 
sliced after 60 
days of storage.
 
 

-






 
 





0
1
2
3
4
5
0
2
4
6
8
10
log CFU/g
 
Storage time (days)
 
(A): 4
o
C
 
a
 
b
 
0
1
2
3
4
5
0
2
4
6
8
10
log CFU/g
 
Storage time (days)
 
(B): 7
o
C
 
CTR
PLSD
PAPD

-
acid
 

-
acid/PAPD
 

-
acid
 

-
acid/PAPD
 
a
 
b
 
77
 
 
The antilisterial activity of hop 

-
acid
 

-
acid
 
at 5 ppm in deli
-
turkey meat was 
different from the results 
in liquid media (Chapter 3).  In liquid media,
 
5 ppm
 

-
acid

-
acid

-
acid
/0.5% PAPD mixtures was listeristatic, while 
5 ppm 

-
acid
 
allowed 
Listeria 
to increase 2.4 logs after 6 days of storage at 7
o
C (Fig. 3.3).  In deli
-
style tu
rkey, 
however, 
Listeria
 
populations 

-
 

-
acid
s 
at 5 
ppm 
after 6 days of storage at 7
o

-
acid

-
acid
/0.5% PAPD mixtures (Fig. 4.3 a
nd 4.4).  These results 
indicate hop acid at 5 ppm alone is not sufficient to inhibit 
Listeria
 
in deli
-
meat while the 
combination of hop acid/PAPD is more effective.
  
These results also support the previous 
findings that 
L. monocytogenes
 
was completely inh
ibited using the combination of 3.0 ppm hop
 

-
acid
, 1.0% potassium lactate, and 0.25% sodium diacetate in broth (Shen and Sofos, 2008).  
However, when used alone, a very high concentration of 
hop
 

-
acid (20,000 ppm) was required 
to reduce the pathogen
 
by 2.1 log CF
U/package 
(Seman et al., 2004).
  
Kr
amer et al. (2014) also 

-
acid
 
extract was 12.5 ppm in broth media and 1000 ppm in the model 
meat marinate.
 

ia and deli
-
meat is 
expected from
 
two reasons:  Firstly, hop
 
acid is 
less mobile in
 
meat batter compared to liquid 
media.  Secondly, hop acid is sequestrated by fat and protein components
 
in meat batter and 
become
 
less 
available to react with 
Listeria
 
membranes.  Being hydrophobic, hop acids can react 
with both mic
robial cell membranes (Schmalreck and Teuber, 1975) and food lipids (Larson et 
al., 1996).  Hence, the antilisterial activity of five hop acids (

-
acid

-
acid
, acid
-
tetra, K
-
tetra, 
and K
-
hexa) 
was assessed with/without PAPD in skim milk and 2% milk (similar fat as deli
-
78
 
 
turkey)
 
in order to investigate if the discrepancy between liquid media and deli
-
meat is due to the 
sequestration of hop acid by f
at in meat batter
. 
 
The average 
L. monocytogenes
 
inoculum 
was 4.2 log CFU/mL for skim milk and 2% 
milk
 
(Fig. 4.5)
.  
Regardless of hop acid type
,
 
0.5% 
PAPD 
and 
0.5%
PAPD
/5 ppm hop acid 
showed 
better inhibition 
in skim milk and TSBYE 
than 
the 
inhibitor
-
free
 
c
ontrols
, with 
inte
rmediate inhibi
tion 
seen 
when the
 
five hop acid extracts
 
were used individually (Fig.
 
4.5).
  
Similarly in 2% milk, 
0.5% PAPD and 0.5% PAPD/5 ppm hop acid showed better inhibition 
than the 
control in 
TSBYE, 
while
 
intermediate inhibition wa
s observed for 
the remaining
 
treatment
s
 
including 
the control in 
2% milk
 
(Fig. 4.5
).
 
 
These results 
clearly 
indicate
 
that the 
addition of hop acids at 
5 ppm is not sufficient 
to inhibit 
Listeria
, 
especially in
 
2% fat milk.  
Larson et al. (1996) observed 
no
 
inhibition
 
difference
s
 
between skim mi
lk 
and skim milk
 
containing 1 and 10 ppm hop 

-
acid
 
during 30 days of storage at 4
o
C, while
 
a 
listericidal 
effect 
was 
seen for 
hop 

-
acid
 
at
 
100 and 1,000 ppm. 
 
 
 
 
79
 
 
 
 
 
 
 
Figure 4
.5
  
L. 
monocytogenes
 
populations
 
in skim milk
 
(A) and 2% milk (B) 
with or without 
diffe
rent hop acid extracts at 5 ppm
 
or
 
0.5
% PAPD during 6 days of storage at 7
o
C. 
 
 

-







 
 





0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
log CFU/mL
 
Storage time (days)
 

-
acid
 

-
acid
 
acid-tetra
K-tetra
K-hexa

-
acid/PAPD
 

-
acid/PAPD
 
acid-tetra/PAPD
K-tetra/PAPD
K-hexa/PAPD
0.5%PAPD
Skim milk
TSBYE
(A):
 
Skim milk
 
abc
 
abc
 
cd
 
cd
 
bc
 
d
 
d
 
d
 
d
 
d
 
d
 
ab
 
a
 
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
log CFU/mL
 
Storage time (days)
 

-
acid
 

-
acid
 
acid-tetra
K-tetra
K-hexa

-
acid/PAPD
 

-
acid/PAPD
 
acid-tetra/PAPD
K-tetra/PAPD
K-hexa/PAPD
0.5%PAPD
2% Milk
TSBYE
(B): 2% milk
 
abc
 
bc
 
abc
 
abc
 
abc
 
c
 
bc
 
bc
 
bc
 
bc
 
bc
 
a
 
a
 
80
 
 
4.4
 
Conclusion
 
Addi
tion of organ
ic aci
ds in processed meats is one common intervention strategy
 
to 
minim
ize
 
Listeria 
growth.  
Although antilisterial activity of hop acids has been 
assessed and 
well 
documented
 
using liquid media
, their use 
in pro
cessed
 
meats has not been 
extensively 
studi
ed
, 
except a few trials using
 
hops solution
 
sprays or dips for processed
 
meats
.  This study indicated 
that hop acids at 5 ppm 
failed
 
to
 
inhibit 
Listeria 
growth 
or induce
 
any synergistic effect
 
with 
0.5% PAPD 
in deli turkey meat and milk during storage of 6 

 
60 days at 4 or 7
o
C
.  Based on 
these findings, 
the addition 
of hop 

-
acid
 
in 
the amount of 4.4 mg/kg (ppm) in
 
co
oked meat and 
5.5 mg/kg (ppm) in
 
casings for meat products (US
-
FDA GRAS Notice Nr 000063) 
(FDA, 2001) 
appears to be insufficient 
to inhibit 
Listeria 
when the 
hop acid
 
is formulated. 
 
 
 
 
 
 
 
 
 
 
 
81
 
 
CHAPTER 
5
: 
ANTILISTE
RIAL EFFECT
 
OF HOP ALPHA AND BETA ACIDS IN 
TURKEY SLURRY AT 7 AND 37
ºC
 
 
5.1
 
Introduction
 
In our previous study (chapters 4), 
single 
addition of 5 ppm 
hop 

-
 

-
acid
 
to d
eli
-
turkey meat was
 
not 
sufficient to 
inhibit 
L
isteria
 
growth with
 
no synergistic effect seen 
with
 
the 
mixture of
 
potassium acetate/potass
ium diacetate (PAPD).  Based on the
se
 
results, t
he next 
question 
to be answered is 
what mini
mum conce
ntration of hop acid is 
for
 
inhibiting
 
L. 
monocytogenes
 
in meat products.  To 
answer the question, turkey slurry was prepar
ed
 
and used
 
as a meat model system
.  
T
he purpose of this study
, therefore,
 
was 
to determine the 
concentrations of 

-
 
and
 

-
acid
s 
required for
 
inhibiting
 
Listeria 
in turkey slurry during 
storage for one day at 37
o
C
 
and 12 days at 7
o
C
.  
The hypothesis of this research is
 
that
 
both 
hop 

-
 
and
 

-
acid
s 
can inhibit 
L. monocytogenes 
at certain 
concentrations 
higher than 5 ppm 
in 
turkey slu
rry du
ring 
storage 
at 7 and 37
o
C
.
 
 
5.2
 
 
Material and methods
 
5.2
.1
 
 
Preparation of hop acids (alpha and beta) and 
L. monocytogenes 
strains
 

-
 

-
acids were obtained from Kalsec Inc
. (Kalamazoo, WI) 
with the 
concentration as shown in Table 3.1.
 
 
For the assessment of antilisterial activity, the hop acid 
extracts were dissolved in 100% ethanol and added to turkey slurries
 
at 0 

 
1000 ppm
.
 
The cocktail of six 
L. monocytogenes 
strains 
was prepared to contain ~1 x 10
8
 
CFU/mL 
and confirmed t
he 
L. mono
cytogenes 
population in the inoculum 
as expla
ined in Chapter 3
. 
 
 
 
82
 
 
5.2
.2
 
Turkey slurries preparation with hop acids 
 
Turkey slurry was  
prepared
 
at the Michigan State University (MSU) Meat l
aboratory 
(East Lansing, MI) by grinding t
urkey
 
breast 
through a 
0
.95
-
cm plate
 
followed by
 
mixing 
th
e 
ground turkey (25%) 
with brine solution (75%), containing 70% water, 2.28% salt, 2.00% sugar, 
0.48% phosphate, and 0.24% nitrite for 1 min in a small food chopper (Model KFC3511, 
KitchenAid, St
. Joseph, MI).  T
he 
slurry 
(100 g) was 
then pasteurized in
 
each of 2
50 mL flasks 
by submerging in a water bath (
85
o
C
)
 
un
til the
 
internal 
temperature
 
of turkey slurry
 
reached to
 
72
o
C
.  To the
 
flask
, 

-
 

-
acid
 
was added
, mixed thoroughly for 2 min with a stir bar, and 
immersed again to the water bath for 3 min
 
to 
sim
ulate the heat exposure 
during cooking
, prior to 
coo
ling to 
37
o
C in icy slurry. 
 
 
5.2
.3
 
Antilisterial activity of hop extracts at 7
 
and 37
o
C
 

-

-
acids were individually dissolved in 1 mL of 100% ethanol and added to 
the 
turkey slurries to achieve 
the 
concentrations of 
0, 
250, 500,
 
750, and 1000 ppm (w/w) prior to
 
incubation at 
37
o
C 
or
 
0, 
5, 25, 50, 100, 
500, and 1000 ppm (w/w)
 
for 
incubation at 
7
o
C
.  Turkey 
slurry without
 
hop acid was 
also 
prepared to serve as a control with the
 
same amount of eth
anol 
used for dissolving 
the 
hop acids.  
T
he 
prepared 
six
-
strain 
L.
 
monocytogenes 
cocktail 
was 
then 
added to each flask 
to achieve
 
ap
proximately 2.0 

 
3.0 log CFU/g
, 
mixed thoroughly, 
and
 
incubated at 37
o
C for 24 h or 7
o
C for 12 days.  Samples were taken initially and after 24 h 
incubation at 37
o
C, or 
initially and 
ev
ery 3 days at 7
o
C.  
Appropriate 
serial dilutions 
in sterile 
phosphate 
buffered saline (PBS) were plated on 
modified Oxford agar (MOX) (Difco, BD) and 
incubated at 
37
o
C
 
for 48 h to enumerate 
L. monocytogenes
 
populations
.  
 
 
83
 
 
5.2
.4
 
Statistical analysis  
 
The microbiological data from triplicate experiments were converted to log
 
CFU/g.  An 
analysis of v
ariance (ANOVA) was performed us
ing the mixed procedure of SAS software (SAS 
Institute, 2002).  
The 
slope of graph of 
Listeria 
population during storage for each treatment 
(growth rate)
 
was calculated
 
to
 
better assess the effect of
 
treatment on 
Listeria 
inhibition
.
 
 

at 
a
 
= 0.05
.
 
 
5.3
 
Results and discussion
 
5
.3
.1
 
 
Antilisterial activity of hop acids at 37
o
C
 
 
Antilisterial activity
 
of
 
0 (Co
ntrol), 250, 500, 750, and 1,000 ppm 
 
hop
 

-
 

-
acid
 
were evaluated 
in turkey slurries
 
after
 
incuba
tion 
at 37
o
C for 24 h
 
(Table 5.1).  
The initial
 
L. 
monocytogenes 
inoculum level 
ranged from 2.22 to 2.40 log CFU/g 
for all treatments 
with no 
signi
ficant diffrence at 0 h at 
37
o
C
 
(
P 
>
 
0.05).  
Cons
idering 
no immediate 
lethal effect 
upon the 
exposure to
 
hop 

-
acids 
in liquid media, this result agrees
 
with the report of Shen and Sofos 
(2008).
  
After incubating 
for 24 h, 
Listeria 
populations 
were 
less than 
the detection limit 
(< 10 
cells/g)
 
at 750 
ppm 

-
acid
 
and 1,000 ppm 

-
acids
,
 
whereas
 
Listeria
 
growth 
using 
500 ppm 

-
 

-
acids
 
was
 
half that of
 
the control
 
(8.02 log CFU/g)
 
(Table 5.1).  
 
In chapter 3, 
Listeria 
popula
tion
s
 
were
 
less than detection  limit 
(< 10 cells/g) 
for
 
50 ppm
 

-
acid
.
 
 
Using
 
25 p
pm
 

-
acid
 
and
 

-
acid, populations were 
reduced to
 
1/8 and half 
of control, 
respectively
,
 
in trypticase soy broth with yeast extract (TSBYE) at 37
o
C for 24 h.
 
 
The 
concentration
 
of 25 ppm 
was 
about 30
 
times
 
lower
 
than the requirement
 
for listericidal activi
ty
 
in 
turkey slurry
.  Larson et al. (1996) 
also 
reported that
 
Listeria
 
growth 
was completely inhibited 
in 
84
 
 
trypticase soy broth containing 10 ppm of hop 

-
 
and 12% 

-
acids) and hop 

-
acids)
 
after 24 h of i
ncubation
 
at 37
o
C
.  In whole milk at 
4
o
C
, however,
 
Listeria 
inhibition 
was seen 
when hop extract
 
III was increased to 
1000 ppm
.  
Again, t
hese findings indicate
 
that the req
uired concentration of hop acid
 
for 
a 
listericidal effect
 
in food
 
is 
3
0 times higher
 
than
 
in liquid media
 
at
 
37
o
C
.  
 
 
Table 5.1
  
Population of 
L. monocytogenes
1
 
(log CFU/g)
 
in turkey slurries 
containing
 
0 to 1000 
ppm 

-
acid
 

-
acid
 
after incubating at 37
o
C for 24 h.  
 
 
Treatment
 
Number of 
L. monocytogenes 
(log CFU/g)
*
 
Time
 
0 h
 
24 h
 

-
acid 250 ppm
 
2.39 ± 0.33 
a
 
4.38 ± 0.42 
cd
 

-
acid 500 ppm
 
2.38 ± 0.34 
a
 
3.96 ± 0.04 
c
 

-
acid 750 ppm
 
2.30 ± 0.30 
a
 
< 1.00 
a
 

-
ac
id 1000 ppm
 
2.22 ± 0.19 
a
 
< 1.00 
a
 

-
acid 250 ppm
 
2.29 ± 0.47 
a
 
4.60 ± 0.18 
d
 

-
acid 500 ppm
 
2.39 ± 0.32 
a
 
4.01 ± 0.06 
c
 

-
acid 750 ppm
 
2.35 ± 0.33 
a
 
1.73 ± 0.24 
b
 

-
acid 1000 ppm
 
2.35 ± 0.34 
a
 
< 1.00 
a
 
0 ppm (Control)
 
2.40 ± 0.42 
a
 
8.02 ± 0.31 
e
 
1
Me
ans 

 
standard deviation of 
n
 
= 6 observations
 
for each reading
.
 
a
-
e
 
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05).
 
*
 
No viable 
L. monocytogenes 
detection 
or the counts below 
minimum detectability of the 
metho
dology (10 cells per gram) 
was marked as < 1.00.
 
 
 
 
 
85
 
 
5
.3
.2
 
 
Antilisterial activity of hop acids at 7
o
C
 
Based on 
our previous resul
ts in liquid med
ia and
 
deli
-
style turkey, antilisterial
 
activities 
of 

-
 

-
acid
s
 
at 0, 25, 50, 100, 500, and 1,000 ppm 
were evaluated 
in turkey slurries
 
duri
ng 
12 days
 
of storage
 
at 
7
o
C
 
(Fig.
 
5.1
).  
B
oth 

-
 
and 

-
acid
s 
were listericidal 
at 
>
 
500 
ppm
, 
indicating
 
that the 
listericidal concentration
 
is 
~
100 times 
higher in 
turkey slurry than
 
in liquid 
media at 
7
o
C.
 
 
Although many 
investigator
s reported that hop acids at 
<
 
10 ppm effectively inhibited the 
growth of 
L. monocytogenes 
in liquid media (Millis and Schendel, 1994; Barney et. al., 1995; 
Larson et. al., 19
96; Shen an
d Sofos, 2008), our work shows
 
that hop acids at < 100 ppm were 
not effective in turkey slurries
 
at 
7
o
C
.
  
In 
accordance
 
with our results, Larson et al. (1996) 
reported that 
a hop acid extract 
containing 

-
acids was listericidal 
at 
1,000 ppm in skim 
milk 
and 2% milk during 35 days of storage at 4
o
C
, with moderate inhibition 
and almost no inhibition at 1
00
 
and 
q
 
10 ppm
, respectively
.  Again, these 
findings indicate
 
that 
the required c
oncentration of hop acids for inhibition of 
Listeria
 
in 
actual 
foods is not 
as
 
same as 
the concentration 
observed 
in liquid media.  
 
 
 
 
 
 
 
 
 
86
 
 
 
Figure 5.1 
 
Population of 
L. 
monocytogenes
 
in 
turkey slurries
 
containing 
0 to 1000 ppm 

-
acid
 
or 

-
acid
 
during 12
 
days of storage at 7
o
C
.
 

-







0
1
2
3
4
5
6
7
8
0
3
6
9
12
log CFU/g
 
Storage time (days)
 

-
acid 5 ppm
 

-
acid 25 ppm
 

-
acid 50 ppm
 

-
acid 100 ppm
 

-
acid 500 ppm
 

-
acid 1000 ppm
 

-
acid 5 ppm
 

-
acid 25 ppm
 

-
acid 50 ppm
 

-
acid 100 ppm
 

-
acid 500 ppm
 

-
acid 1000 ppm
 
Control
a
 
ab
 
b
 
c
 
cd
 
cd
 
ab
 
ab
 
ab
 
ab
 
cd
 
d
 
a
 
87
 
 
In foods, 
hop
 
acid
 
is not a popular food ingredient 
due to 
the 
undesirable 
bitter taste.  
Although our
 
results indicated that hop 

-
 
and 

-
acid
s
 
at 
> 500 ppm
 
are unlikely added to 
food
s, 
hop acids could be used in 
combination with organic acids to reduce both 
the 
sour 
taste from 
organic acids 
and 
t
he bitter flavor from hop acids.
  
In sensory evaluation
s
, 
ne
gative odor was 
detected in 
ham containing 0.
2% sodium diacetate
 
(Stekelenburg and Kant
-
Muermans, 2001) 
and bitter taste at > 50 ppm 
for
 
purified 
hop 

-
acid
 
(
Millis et al., 1994)
.
 
  
In our previous study 
(chapter 3), 
addition of 
1% 
PAPD (80% potassium acetate/20% 
potassium diacetate) 
containing 0.2% potassium diacetate 
resulted in 
lower flavor 
and overall 
acceptability
 
scores
 
in low
-
sodium frankfurters 
compared to
 
the 
inhibitor
-
free 
control (Sansawat 
et al., 2013).  As a result, it 
will be interesting if the
 
combination of
 
PAPD at < 1%
 
and hop acid 
at < 50 ppm 
could minimize both 
sour 
and
 
bitter flavor
 
while maintaining the 
Listeria 
inhibition
.  
 
Based o
n these
 
findings, the USDA allowance for
 
hop acids 
used 
meat products 
(4.4 
mg/kg in cooked meats and 5.5 mg/kg in meat product in casings) 
(US/
FDA GRAS Notice Nr 
000063
)
 
(FDA, 2001)
 
need
s to be updated 
for 
practical application in case of
 
formulation to me
at 
batter
.
 
I
n liquid media
, hop acid
 
can
 
direct
ly
 
contact
 
L. monocytogenes
 
and result 
in
 
effe
ctive 
inhibition
, which is not
 
true in 
meat batter
s
.  In our study, the minimal inhibitory concentration 
of hop 

-
 

-
acid
 
was between 6.3 and 3.1 ppm in li
quid
 
media (Table 3
.4
, Chapter 3), but no 
inhibition was observed in deli
-
turkey, skim milk, and 2% milk 
at
 
5 ppm
 
regardless of 
hop 

-
 
or 

-
acid
 
(Fig.
 
4.2, 4.5, 4.6 Chapter 
4).  It is known that increased 
hydrophobicity of hop acid leads 
to 
greater antimicrobi
al activity 
due to 
increased 
interaction
 
with 
the 
bacteria
l cell membrane 
(Etoh et al., 1994; Schmalreck et al., 
1975).  
R
educed activity of hop ext
ract 
in food
 
is expected
 
from
 
the sequestration 
of hydrophobic groups 
by
 
food lipid
s (Larson et al., 1996)
. 
 
As a result,
 
88
 
 
enc
apsulation
 
of hop acids would be additional solution
 
s
o that 
the encapsulated hop acids can be 
less sequestrated 
during 
batter 
mixing and 
effectively released after 
emulsifying fats and 
coagulating proteins 
during cooking
.
89
 
 
5.4
 
Conclusion
 
H
op 

-
 

-
acid
s exhibited antilisterial activity in turkey slurries 
at 
the 
concentration
s
 
>
 
750 ppm 
during storage at 3
7
o
C
 
for 24 h or at 
the 
concentration
s
 
>
 
500 ppm at 
7
o
C
 
for 12 days.  
However, 
the high concentration of hop acids might be not practica
l due to 
the prediction of 
negative sensory impacts.  Two potential solutions for the implementation of hop acids could be: 
(1) combination with 
an 
organic acid 
at 
the 
levels below the threshold for
 
bitter 
and sour taste,
 
and
 
(2) encapsulation of the hop a
cids 
to avoid
 
any 
s
equestration 
by food components 
during 
batter mixing 
and 
maintain antilisterial activity
 
with no sensory issue
. 
90
 
 
SUMMARY
 
 
Continuous
 
outbreaks of listeriosis 
urged 
food processors to find a
 
better 
way to control 
L.
 
monocytogenes 
especial
ly in
 
ready
-
to
-
eat (RTE) meat products
.  
Formulation 
of antimicrobial 
agents in food
s is 
one of common methods 
to
 
control
 
bacteria including
 
L.
 
monocytogenes.
  
Regardless of 
surface application or formulation, 
development 
of 
a new
 
antilisterial agent 
and 
i
nnovative
 
combinations 
of pre
-
existing additives
 
are always desirable 
for
 
pathogen inhibition 
and 
improve
d
 
product quality
.  
Results
 
from this research 
indicated that 
the
 
mixtures
 
of hop 
acid
s and
 
organic acid
 
salt
s
 
were more effective than any single appl
ication
 
on
 
Listeria 
inhibition 
in liquid 
media
.
  
Concerning
 
Listeria 
inhibition and product sensory quality,
 
the
 
mixture
 
of
 
potassium acetate and potassium diacetate
 
(
PAPD)
 
out of 9 organic acids/mixtures was 
the most 
effective in 
Listeria 
inhibition
 
but n
ot for eating quality 
of frankfurter in low
-
sodium.  In case of 
hop acids,
 
ad
dition of 

-
 

-
acid
 
out of 8 hop acids
 
was most effective in 
Listeria 
inhibit
ion in 
liquid me
dia, but the effectiveness was not the same in meat paste.  Upon mixing 
the best 
organic acid and 
one of the two best 
hop ac
id
s, they
 
induced synergistic 
effects on 
Liste
ria
 
inhibition in liquid media, but not in meat paste
.
 
 
These results are expected from the 
sequestration of hop acids by lipids and proteins in meat batter.
  
Therefore, 
it will
 
be interesting 
to 
conduct an additional study to find the right 
levels of hop 
acid and 
PAPD 
in mixture 
that 
can 
provide 
effective 
Listeria 
inhibition with
 
no sensory quality
 
loss
.
  
More interestingly, 
encapsulaiton of hop acids will be desirable if it can prevent any sequestration of hop acids 
during meat batter preparation.
 
  
 
 
 
 
91
 
 
F
UTURE RECOMMENDATIONS
 
 
R
esults of
 
this disse
rtation
 
indicated that
 
the combination of 
organic acids 
and hop acids 
possess the potential 
to
 
improve
 
antilisterial activity and sensory attributes.  When incubating 
Listeria 
with 0.5% PAPD and hop
 

-
 
or 

-
acid 
at 25 ppm, the pathogen populatio
ns decreased 
from 5.7 log CFU/mL
 
to non
-
de
tectable level in liquid media.  
In 
single
 
addition, 
the required 
hop acid for 
Listeria
 
inhibition was 500 ppm in meat batter, whereas the minimum inhibitory 
activity of hop acid wa
s betwe
en 3.1 

 
6.3 ppm
 
in liquid media
.  
Our results also indicate that 
when formulated 
hop acids to 
deli
-
style turkey meats
 
at the allowance of USDA
-
FSIS, the hop 
acids
 
did not inhibit the growth of 
L. monocytogenes 
nor generate further
 
inhibition 
upon 
c
ombining 
with PAPD
.
  
Therefore, 
these results suggest that USDA
-
FSIS should 
reconsider the
 
allowance 
level 
in 
case of formulation
.  I
n future studies, 
it wi
ll be interesting to find 
that if 
formulation of 
encapsulated hop acids can 
provide better 
inhibit
io
n on
 
L. monocytogenes
 
in meat 
paste with 
no sensory issue.  
It will be also interesting 
to know 
that 
if 
m
ixture
 
of 
encapsulated 
hop acid
s
 
with PAPD
 
or 
other antimicrobial agents
 
can 
generate synergistic effects
 
in
 
deli
-
style 
t
urkey.
  
Given no inhibition in the range of sensory issue (< 25 ppm
 
hop acids
), we could suggest 
that hop acids may not 
be an adequate inhibitor in processed meat products.
  
Providing better 
inhibition
 
and synergistic effects with other inhibitors upon encapsulation, the hop acids could be 
useful as a natural food preservative.
 
 
 
 
 
 
92
 
 
 
 
 
 
 
 
 
 
 
 
APPENDICES
 
 
 
 
 
 
 
 
 
 
 
 
 
 
93
 
 
APPENDIX A
 
 
Tables of supplemental data
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
94
 
 
Table 
A
.
1
 
Population
 
of 
L. monocytogenes
1,2,3
 
on vacuum
-
packaged 
full
-
sodium 
frankfurters 
with powdered
 
or liquid inhibitors
4
 
during 90 days of storage at 4, 7 and 10ºC.
 
 
 
1
Mean values with same letters in the same column were not signific
antly different (
P
 

 
0.05).     
 
2
Means 

 
standard deviation of 
n
 
= 6 observations
 
for each reading, except for control
. 
 
3
The minimum detectability of the methodology was > 10 cells per gram. 
 
4
Inhibitors 
in the formulation
 
as in Table 
2.
3
 
Treatments
 
0
 
15
 
30
 
45
 
60
 
75
 
90
 
Storage at 4
o
C
 
CTR (0%)
 
4
.57
 
± 
0.17
a
 
5.88 ± 0.18
a
 
7.10 ± 0.32
a
 
7.12 ± 1.49
a
 
7.75 ± 0.31
a
 
7.70 ± 0.19
a
 
7.56 ± 0.41
a
 
PI
-
I (0.5%)
 
4.56 ± 0.25
a
 
4.58 ± 0.15
b
 
4.77 ± 0.39
b
 
5.31 ± 1.15
b
 
5.76 ± 1.27
b
 
6.23 ± 0.78
b
 
6.90 ± 0.44
ab
 
PI
-
2 (1.00%)
 
4.52 ± 0.17
a
 
4.29 ± 0.26
b
 
4.26 ± 0.01
bc
 
4.30 ± 
0.33
b
 
4.54 ± 0.46
bc
 
4.35 ± 0.40
cd
 
4.70 ± 0.80
cd
 
PI
-
3 (0.65%)
 
4.58 ± 0.54
a
 
4.28 ± 0.17
b
 
4.21 ± 0.02
bc
 
4.18 ± 0.15
b
 
4.30 ± 0.16
bc
 
4.13 ± 0.11
cd
 
4.13 ± 0.10
d
 
PI
-
4 (0.75%)
 
4.57 ± 0.26
a
 
4.26 ± 0.22
b
 
4.22 ± 0.12
bc
 
4.20 ± 0.27
b
 
4.11 ± 0.13
c
 
4.05 ± 0.15
d
 
4.02 ± 
0.22
d
 
PI
-
5 (1.00%)
 
4.60 ± 0.34
a
 
4.19 ± 0.23
b
 
4.10 ± 0.22
c
 
4.11 ± 0.36
b
 
4.09 ± 0.18
c
 
3.94 ± 0.24
d
 
4.06 ± 0.31
d
 
LI
-
1 (2.5%)
 
4.52 ± 0.3
9
a
 
4.26 ± 0.27
b
 
4.32 ± 0.17
bc
 
4.60 ± 0.58
b
 
5.31 ± 0.98
bc
 
5.39 ± 1.19
bc
 
5.88 ± 1.08
bc
 
LI
-
2 (2.5%)
 
4.56 ± 0.47
a
 
4.18 ± 0.23
b
 
4.12 ± 0.16
c
 
4.11 ± 0.24
b
 
4.18 ± 0.41
bc
 
4.18 ± 0.32
cd
 
4.23 ± 0.52
d
 
LI
-
3 (2.5%)
 
4.57 ± 0.53
a
 
4.26 ± 0.31
b
 
4.23 ± 0.05
bc
 
4.68 ± 0.83
b
 
5.25 ± 1.35
bc
 
5.05 ± 0.78
bcd
 
5.77 ± 0.94
bc
 
LI
-
4 (2.5%
)
 
4.59 ± 0.24
a
 
4.31 ± 0.21
b
 
4.17 ± 0.10
c
 
4.37 ± 0.28
b
 
4.69 ± 0.56
bc
 
4.55 ± 0.74
cd
 
4.54 ± 0.69
cd
 
Storage at 7
o
C
 
CTR (0%)
 
4.57
 
± 
0.17
a
 
6.99 ± 0.44
a
 
7.56
 
± 0.25
a
 
7.73 ± 0.51
a
 
7.49 ± 0.38
a
 
7.32 ± 0.44
a
 
7.15 ± 0.29
ab
 
PI
-
I (0.5%)
 
4.56 ± 0.25
a
 
5.77 ± 0.64
b
 
6.92 ± 0.25
b
 
7.10 ± 0.54
a
 
7.07 ± 0.66
ab
 
7.62 ± 0.09
a
 
7.78 ± 0.05
a
 
PI
-
2 (1.00%)
 
4.52 ± 0.17
a
 
4.26 ± 0.25
d
 
4.63 ± 0.44
cd
 
5.39 ± 0.77
cd
 
5.26 ± 0.97
cde
 
6.19 ± 0.56
abc
 
6.79 ± 0.57
abc
 
PI
-
3 (0.65%)
 
4.58 ± 0.54
a
 
4.24 ± 0.17
d
 
4.19 ± 0.18
d
 
4.55 ± 0.54
de
 
4.61 ± 1.04
de
 
4.77 ± 0.89
cd
 
6.13 ± 0.62
bc
 
PI
-
4 (0.75%)
 
4.57 ± 0.26
a
 
4.28 ± 0.14
d
 
4.32 ± 0.34
d
 
4.43 ± 0.39
de
 
3.98 ± 0.07
e
 
4.10 ± 0.32
d
 
4.10 ± 0.46
d
 
PI
-
5 (1.00%)
 
4.60 ± 0.34
a
 
4.22 ± 0.17
d
 
4.17 ± 0.28
d
 
4.23 ± 0.32
e
 
4.09 ± 0.10
e
 
4.05 ± 0.38
d
 
4.41 ± 0.74
d
 
LI
-
1 (2.5%)
 
4.52 ± 0.3
9
a
 
5.12 ± 0.24
bc
 
5.20 ± 0.74
c
 
6.74 ± 0.65
ab
 
6.56 ± 0.63
abc
 
7
.12 ± 0.38
ab
 
7.28 ± 0.09
ab
 
LI
-
2 (2.5%)
 
4.56 ± 0.47
a
 
4.18 ± 0.29
d
 
4.53 ± 0.56
cd
 
4.94 ± 1.24
cde
 
4.78 ± 0.98
de
 
5.31 ± 1.41
bcd
 
5.73 ± 0.58
c
 
LI
-
3 (2.5%)
 
4.57 ± 0.53
a
 
4.76 ± 0.40
cd
 
5.23 ± 0.74
c
 
5.96 ± 0.95
bc
 
5.83 ± 0.78
bcd
 
6.28 ± 0.96
abc
 
7.18 ± 0.45
ab
 
LI
-
4 (2
.5%
)
 
4.59 ± 0.24
a
 
4.50 ± 0.22
cd
 
4.58 ± 0.27
cd
 
5.22 ± 0.34
cde
 
5.47 ± 0.98
bcde
 
5.80 ± 0.52
abcd
 
6.46 ± 0.33
bc
 
Storage at 10
o
C
 
CTR (0%)
 
4.57
 
± 
0.17
a
 
7.
2
4
 
± 0.
2
0
a
 
7.
48
 
± 0.
 
35
a
 
7.3
0
 
± 0.
4
5
a
 
6.
62
 
± 0.6
4
abc
 
6.15
 
± 0.
79
ab
 
5.
34
 
± 1.
43
abc
 
PI
-
I (0.5%)
 
4.56 ± 0.25
a
 
7.
35
 
± 0.
25
a
b
 
7.5
6
 
± 0.2
2
a
 
7.
33
 
± 0.2
3
a
 
7.
08
 
± 0.
20
ab
 
6.
91
 
± 0.
66
ab
 
6.
8
1
 
± 0.
49
a
 
PI
-
2 (1.00%)
 
4.52 ± 0.17
a
 
6.
0
1
 
± 0.
23
c
d
 
7.
1
9 ± 0.
25
a
b
 
7.
51
 
± 0.
06
a
 
7.
50
 
± 0.
13
a
 
7.
18
 
± 0.
23
a
 
 
7
.86
 
± 0.
29
a
 
PI
-
3 (0.65%)
 
4.58 ± 0.54
a
 
5.
49
 
± 0.
30
de
 
6.59
 
± 0.
71
a
bc
 
6.90
 
± 0.
1
1
a
 
6.62
 
±
0.08
a
bc
 
6.58
± 0.
22
a
b
 
6.59
 
± 
0.39
a
b
 
PI
-
4 (0.75%)
 
4.57 ± 0.26
a
 
4.64
 
± 0.46
e
 
5.44
 
± 0.95
cd
 
5.33
 
± 0.67
b
 
5.1
0 ± 0.24
bc
 
4.65
 
± 0.31
b
 
4.64
 
± 0.02
bc
 
PI
-
5 (1.00%)
 
4.60 ± 0.34
a
 
4.76
 
± 0.14
e
 
4.89
 
± 1.03
d
 
5.08
 
± 0.48
b
 
5.01
 
± 0.32
c
 
4.74
 
± 0.41
b
 
4.03
 
± 0.27
c
 
LI
-
1 (2.5%)
 
4.52 ± 0.3
9
a
 
6.91 ± 0.20
ab
c
 
7.60
 
± 0.23
a
 
7.48
 
± 0.09
a
 
7.42
 
± 0.14
a
 
7.17
 
± 0.25
a
 
7.23
 
± 0.65
a
 
LI
-
2 (2.5%)
 
4.56 ± 0.47
a
 
5.12
 
± 0.69
de
 
6.16 ± 0.2
a
bcd
 
6.31
 
± 0.27
a
b
 
6.39
 
± 0.33
ab
c
 
6.20
 
± 0.56
ab
 
6.20
 
± 0.83
ab
 
LI
-
3 (2.5%)
 
4.57 ± 0.53
a
 
6.80
 
± 0.25
abc
 
6.88
 
± 0.40
a
bc
 
6.98 ± 0.0
3
a
 
6.79
 
± 0.25
abc
 
6.81
 
± 0.20
a
b
 
6.69
 
± 0.22
ab
 
LI
-
4 (2.5%
)
 
4.59 ± 0.24
a
 
6.26
 
± 0.13
bc
d
 
5.90
 
± 0.17
bcd
 
7.03
 
± 0.06
a
 
6.16
 
± 0.11
abc
 
5.12
 
± 0.29
a
b
 
5.17
 
± 0.19
a
bc
 
95
 
 
Table 
A
.
2
 
Area
1
 
under graph 
of 
L. monocytogenes 
 
population 
on vacuum
-
packaged 
full
-
sodium 
frankfurters with powdered
 
or liquid inhibitors
2
 
during 90 days of storage at 4, 7 and 
10ºC.
 
 
 
1
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05).     
 
2
Inhibitors 
in the formulation
 
as in Table 
2.
3
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Treatments
 
Area under graph 
 
4
ºC
 
7
ºC
 
1
0
ºC
 
CTR (0%)
 
623.37 
a
 
649.77 
a
 
606.82 
a
 
PI
-
I (0.5%)
 
484.94 
b
 
609.01 
ab
 
630.24 
a
 
PI
-
2 (1.00%)
 
394.88 
bc
 
470.44 
cde
 
618.35 
a
 
PI
-
3 (0.65%)
 
380.91
c
 
414.92 
de
 
568.25 
a
 
PI
-
4 (0.75%)
 
376.30 
c
 
380.87 
e
 
448.09 
b
 
PI
-
5 (1.00%)
 
370.27 
c
 
377.82 
e
 
433.90 
b
 
LI
-
1 (2.5%)
 
435.78 
bc
 
549.16 
abc
 
641.97 
a
 
LI
-
2 (2.
5%)
 
376.76 
c
 
432.48 
de
 
535.09 
ab
 
LI
-
3 (2.5%)
 
428.92 
bc
 
508.36 
b
cd
 
599.99 
a
 
LI
-
4 (2.5%)
 
398.79 
bc
 
465.57 
cde
 
532.26 
ab
 
 
 
 
 
96
 
 
Table
 
A
.
3
 
Population
 
of 
L. monocytogenes
1,2,3 
on vacuum
-
packaged 
low
-
sodium 
frankfurters 
 
     
with powdered
 
or liquid inhibitors
4
 
during 90 days of storage at 4, 7 and 10ºC.
 
 
Treatments
 
0
 
15
 
30
 
45
 
60
 
75
 
90
 
Stor
age at 4
o
C
 
CTR (0%)
 
4.74 ± 0.12
a
 
6.28 ± 0.87
a
 
6.65 ± 1.45
a
 
7.26 ± 0.93
a
 
7.37 ± 0.44
a
 
7.25 ± 0.88
a
 
6.96 ± 0.84
a
 
 
PI
-
2 (1.00%)
 
4.69 ± 0.19
a
 
4.69 ± 0.25
b
 
5.16 ± 0.59
ab
 
5.34 ± 0.98
bc
 
5.95 ± 0.88
b
 
6.15 ± 1.82
ab
 
6.51 ± 0.95
a
 
PI
-
3 (0.65%)
 
4.79 ± 0.04
a
 
4.52 ± 0
.21
b
 
4.69 ± 0.24
b
 
4.91 ± 0.61
bc
 
4.94 ± 0.30
bc
 
5.29 ± 0.90
b
 
5.36 ± 1.14
ab
 
PI
-
5 (1.00%)
 
4.70 ± 0.07
a
 
4.35 ± 0.17
b
 
4.34 ± 0.05
b
 
4.45 ± 0.19
c
 
4.21 ± 0.09
c
 
4.24 ± 0.08
b
 
4.15 ± 0.46
b
 
LI
-
4 (2.5%
)
 
4.71 ± 0.08
a
 
4.81 ± 0.36
b
 
5.17 ± 0.82
ab
 
5.81 ± 0.93
b
 
6.11 ± 0.93
b
 
5.77 ± 1.46
ab
 
5.96 ± 1.75
a
 
Storage at 7
o
C
 
CTR (0%)
 
4.74 ± 0.12
a
 
7.39 ± 0.56
a
 
7.38 ± 0.46
a
 
7.60 ± 0.30
a
 
7.54 ± 0.05
a
 
7.29 ± 0.10
a
 
7.16 ± 0.63
a
 
PI
-
2 (1.00%)
 
4.69 ± 0.19
a
 
5.32 ± 0.55
b
 
6.27 ± 0.87
ab
 
7.10 ± 0.67
a
 
7.15 ± 0.64
a
 
7.53 ± 0.39
a
 
7.36 ± 0.57
a
 
PI
-
3
 
(0.65%)
 
4.79 ± 0.04
a
 
4.73 ± 0.37
bc
 
5.28 ± 0.59
bc
 
5.92 ± 0.76
ab
 
6.38 ± 0.68
a
 
6.47 ± 0.58
a
 
6.13 ± 0.49
b
 
PI
-
5 (1.00%)
 
4.70 ± 0.07
a
 
4.34 ± 0.20
c
 
4.30 ± 0.14
c
 
4.30 ± 0.13
b
 
4.25 ± 0.12
b
 
4.53 ± 0.46
b
 
4.46 ± 0.63
c
 
LI
-
4 (2.5%
)
 
4.71 ± 0.08
a
 
5.31 ± 0.79
b
 
6.01 ± 1.
33
ab
 
6.37 ± 1.43
a
 
6.50 ± 1.00
a
 
6.83 ± 1.00
a
 
6.91 ± 0.44
a
 
Storage at 10
o
C
 
CTR (0%)
 
4.74 ± 0.12
a
 
7.60 ± 0.31
a
 
7.55 ± 0.09
a
 
7.02 ± 0.38
ab
 
6.56 ± 0.46
a
 
5.69 ± 0.72
ab
 
5.77 ± 0.58
ab
 
PI
-
2 (1.00%)
 
4.69 ± 0.19
a
 
6.86 ± 0.24
ab
 
7.62 ± 0.47
a
 
7.70 ± 0.12
a
 
7.21 ± 0.52
a
 
7.01 ± 0.30
a
 
6.98 ± 0.37
a
 
PI
-
3 (0.65%)
 
4.79 ± 0.04
a
 
5.55 ± 0.75
cd
 
7.11 ± 0.17
ab
 
7.03 ± 0.62
ab
 
6.69 ± 0.64
a
 
6.12 ± 1.50
ab
 
5.89 ±1.07
ab
 
PI
-
5 (1.00%)
 
4.70 ± 0.07
a
 
4.48 ± 0.13
d
 
4.49 ± 0.25
c
 
4.67 ± 0.50
c
 
4.81 ± 1.25
b
 
4.61 ± 1.13
b
 
4.72 ± 0.12
b
 
LI
-
4 (2.5%
)
 
4
.71 ± 0.08
a
 
6.32 ± 1.04
bc
 
6.82 ± 0.53
b
 
6.50 ± 0.95
b
 
6.57 ± 0.67
a
 
6.06 ± 1.29
ab
 
6.16 ± 1.38
ab
 
1
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05). 
2
M
eans
 

 
standard deviation
 
of 
n
 
= 6 observations
 
for each reading
,
 
except for control
. 
 
3
The minimum detectability of the methodology was > 10 cells per gram.
 
4
Inhibitors 
in the formulation
 
as in Table 
2.
3
 
 
 
Table 
A
.
4
 
Area
1
 
under graph 
of 
L. monocytogenes 
 
population 
on vacuum
-
packaged 
low
-
sodium 
frankfurters with powder
ed
 
or liquid inhibitors
2
 
during 90 days of storage at 4, 7 and 10ºC.
 
 
 
1
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05).     
 
2
Inhibitors 
in the formulation
 
as in Table 
2.
3
 
 
 
Treatments
 
Area under graph 
 
4
ºC
 
7
ºC
 
1
0
ºC
 
CTR (0%)
 
609.77 
a
 
647.35 
a
 
595.08 
a
 
PI
-
2 (1.00%)
 
493.36 
a
b
 
590.99 
a
 
633.57 
a
 
PI
-
3 (0.65%)
 
441.32 
b
 
513.50 
ab
 
567.72 
a
 
PI
-
5 (1.00%)
 
390.18
 
b
 
394.36 
b
 
416.55 
b
 
LI
-
4 (2.5%)
 
495.14 
a
b
 
552.65 
a
 
565.67 
a
 
 
 
 
 
97
 
 
Table 
A
.
5
 
Population 
of mesophilic aero
bic bacteria
1,2,3
 
on vacuum
-
packaged frankfurters with 
powdered
 
or liquid inhibitors
4
 
during 90 days of storage at 4, 7 and 10ºC.
 
1
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05).     
 
2
M
eans
 

 
standard deviation
 
of 
n
 
= 6 observations
 
for each reading
, ex
cept for control
. 
 
3
The minimum detectability of the methodology was > 10 cells per gram.
 
4
Inhibitors 
in the formulation
 
as in Table 
2.
3
 
 
 
Treatments
 
0
 
15
 
30
 
45
 
60
 
75
 
90
 
Storage at 4
o
C
 
CTR (0%)
 
1.40
 
± 0.96
a
 
3.04 ± 2.33
a
 
6.57 ± 0.29
a
 
7.50 ± 0.11
a
 
6.98 ± 0.83
a
 
6.74
 
± 1.50
a
 
7.07 ± 1.26
a
 
PI
-
I (0.5%)
 
1.56 ± 0.50
a
 
1.83 ± 1.69
a
 
3.00 ± 1.60
b
 
5.71 ± 0.66
b
 
6.00 ± 0.78
ab
 
5.15 ± 1.91
ab
 
6.26 ± 2.48
ab
 
PI
-
2 (1.00%)
 
1.64 ± 0.18
a
 
1.54 ± 0.99
a
 
2.54 ± 1.25
b
 
5.55 ± 0.74
b
 
5.32 ± 0.29
b
 
4.82 ± 0.49
ab
 
5.05 ± 1.66
ab
 
PI
-
3 (0.65%)
 
1.91 ±
 
0.55
a
 
1.69 ± 1.03
a
 
2.98 ± 0.85
b
 
5.60 ± 0.72
b
 
5.27 ± 0.28
b
 
5.47 ± 0.92
ab
 
4.74 ± 1.65
b
 
PI
-
4 (0.75%)
 
1.43
 
± 0.96
a
 
1.48 ± 0.56
a
 
2.88 ± 0.87
b
 
5.36 ± 0.89
b
 
5.35 ± 0.35
b
 
4.91 ± 1.07
ab
 
4.49 ± 1.44
b
 
PI
-
5 (1.00%)
 
1.25 ± 0.3
6
a
 
1.61 ± 0.99
a
 
2.05 ± 0.91
b
 
5.27 ± 0.77
b
 
4.69 ± 0.70
b
 
4.06 ± 0.51
b
 
5.14 ± 1.19
ab
 
LI
-
1 (2.5%)
 
1.42 ± 0.39
a
 
2.05 ± 1.19
a
 
2.76 ± 0.75
b
 
5.69 ± 0.67
b
 
6.03 ± 0.82
ab
 
5.86 ± 1.47
ab
 
6.12 ± 1.77
ab
 
LI
-
2 (2.5%)
 
1.77 ± 0.47
a
 
1.39 ± 1.33
a
 
2.17 ± 0.83
b
 
4.74 ± 0.58
b
 
5.12 ± 0.38
b
 
4.90 ± 1.58
ab
 
5.39 ± 1.60
ab
 
LI
-
3 (2.5%)
 
1.77 ± 0.53
a
 
1.36 ± 0.58
a
 
3.12 ± 1.84
b
 
5.82 ± 0.58
b
 
5.71 ± 0.43
ab
 
5.14 ± 1.19
ab
 
6.53 ± 0.79
ab
 
LI
-
4 (2.5%
)
 
1.66 ± 0.25
a
 
1.73 ± 0.25
a
 
2.74 ± 0.76
b
 
5.51 ± 0.73
b
 
5.74 ± 0.55
ab
 
5.35 ± 1.05
ab
 
5.79 ± 0.86
ab
 
Storage at 7
o
C
 
CTR (0%)
 
1.40
 
± 0.96
a
 
4.17
 
± 3.40
a
 
6.74 ± 0.29
a
 
7.60 ± 0.08
a
 
6.78 ± 0.84
a
 
6.64 ± 0.95
a
 
6.36 ± 1.85
ab
 
PI
-
I (0.5%)
 
1.56 ± 0.50
a
 
1.73 ± 2.16
ab
 
3.47 ± 1.19
b
 
6.13 ± 0.40
ab
 
6.65 ± 0.61
a
 
5.64 ± 1.13
ab
 
6.19 ± 1.82
ab
 
PI
-
2 (1.00%)
 
1.64 ± 0.18
a
 
1.36 ± 1.11
b
 
3.53 ± 1.06
b
 
5.81 ± 0.25
b
 
5.25 ± 
0.41
b
 
4.92 ± 0.57
ab
 
5.12 ± 0.94
b
 
PI
-
3 (0.65%)
 
1.91 ± 0.55
a
 
2.31 ± 1.70
ab
 
3.40 ± 1.06
b
 
5.62 ± 0.49
b
 
5.91 ± 0.69
ab
 
5.17 ± 0.71
ab
 
5.83 ± 1.27
ab
 
PI
-
4 (0.75%)
 
1.43
 
± 0.96
a
 
2.09 ± 1.88
ab
 
3.75 ± 0.75
b
 
5.53 ± 0.61
b
 
5.72 ± 0.58
ab
 
5.23 ± 1.44
ab
 
5.18 ± 1.02
b
 
PI
-
5 
(1.00%)
 
1.25 ± 0.3
6
a
 
1.43 ± 1.79
b
 
3.62 ± 0.85
b
 
5.50 ± 0.62
b
 
5.41 ± 0.37
b
 
4.57 ± 0.54
b
 
5.20 ± 0.87
ab
 
LI
-
1 (2.5%)
 
1.42 ± 0.39
a
 
2.21 ± 1.65
ab
 
4.09 ± 0.85
b
 
6.21 ± 0.51
ab
 
6.36 ± 0.47
ab
 
5.95 ± 1.51
ab
 
6.50 ± 1.40
ab
 
LI
-
2 (2.5%)
 
1.77 ± 0.47
a
 
1.98 ± 1.53
ab
 
4.19 ± 
0.59
b
 
5.65 ± 0.87
b
 
5.93 ± 0.77
ab
 
5.89 ± 1.19
ab
 
5.85 ± 1.47
ab
 
LI
-
3 (2.5%)
 
1.77 ± 0.53
a
 
2.52 ± 1.85
ab
 
4.61 ± 1.21
ab
 
6.48 ± 0.63
ab
 
6.30 ± 0.81
ab
 
6.22 ± 1.23
ab
 
6.58 ± 1.17
a
 
LI
-
4 (2.5%
)
 
1.66 ± 0.25
a
 
2.12 ± 1.52
ab
 
4.41 ± 1.14
b
 
6.34 ± 0.46
ab
 
6.27 ± 0.67
ab
 
6.50 
± 0.45
a
 
6.27 ± 1.25
ab
 
Storage at 10
o
C
 
CTR (0%)
 
1.40
 
± 0.96
a
 
4.22 ± 3.42
a
 
7.63 ± 0.58
a
 
7.50 ± 0.30
abc
 
7.06 ± 1.03
a
 
7.59 ± 0.36
a
 
7.20 ± 0.78
a
 
PI
-
I (0.5%)
 
1.56 ± 0.50
a
 
3.58 ± 3.13
a
 
7.07 ± 0.19
abc
 
7.64 ± 0.33
ab
 
7.07 ± 0.94
a
 
6.92 ± 1.07
a
 
7.44 ± 0.67
a
 
PI
-
2 (
1.00%)
 
1.64 ± 0.18
a
 
3.38 ± 3.25
a
 
6.41 ± 0.78
bcd
 
7.54 ± 0.07
abc
 
7.07 ± 0.81
a
 
6.64 ± 1.60
a
 
7.06 ± 0.92
a
 
PI
-
3 (0.65%)
 
1.91 ± 0.55
a
 
3.50 ± 2.77
a
 
7.17 ± 0.14
ab
 
7.37 ± 0.26
abc
 
6.86 ± 1.10
a
 
6.63 ± 2.18
a
 
7.17 ± 0.48
a
 
PI
-
4 (0.75%)
 
1.43
 
± 0.96
a
 
3.61 ± 3.12
a
 
6.09 ±
 
0.44
bcd
 
6.78 ± 0.39
bcd
 
6.27 ± 0.60
a
 
7.14 ± 0.92
a
 
7.10 ± 0.25
a
 
PI
-
5 (1.00%)
 
1.25 ± 0.3
6
a
 
2.75 ± 2.07
a
 
5.66 ± 0.52
d
 
6.31 ± 0.24
d
 
6.09 ± 1.04
a
 
6.34 ± 1.52
a
 
7.03 ± 0.27
a
 
LI
-
1 (2.5%)
 
1.42 ± 0.39
a
 
3.95 ± 3.00
a
 
7.00 ± 0.34
abc
 
7.61 ± 0.37
abc
 
6.78 ± 1.13
a
 
7.35 ±
 
0.20
a
 
7.21 ± 0.79
a
 
LI
-
2 (2.5%)
 
1.77 ± 0.47
a
 
3.39 ± 2.77
a
 
5.99 ± 0.57
cd
 
6.69 ± 0.05
cd
 
6.21 ± 0.94
a
 
6.25 ± 0.77
a
 
7.01 ± 0.11
a
 
LI
-
3 (2.5%)
 
1.77 ± 0.53
a
 
3.76 ± 3.15
a
 
6.91 ± 0.34
abc
 
7.72 ± 0.40
a
 
7.17 ± 0.69
a
 
7.30 ± 0.18
a
 
7.23 ± 0.27
a
 
LI
-
4 (2.5%
)
 
1.66 ± 0.25
a
 
3.90 ± 3.18
a
 
6.73 ± 0.34
abcd
 
7.51 ± 0.50
abc
 
6.75 ± 1.02
a
 
6.70 ± 1.09
a
 
7.07 ± 0.30
a
 
98
 
 
Table 
A
.
6
 
 
Population
 
of mesophilic aerobic bacteria
1,2,3
 
on vacuum
-
packaged 
low
-
sodium 
 
      
 
frankfurters
 
with pow
dered
 
or liquid 
Listeria
 
growth inhibitors
4
 
during 90 days of 
storage at 4, 7 and 10ºC.
 
 
Treatments
 
0
 
15
 
30
 
45
 
60
 
75
 
90
 
Storage at 4
o
C
 
CTR (0%)
 
2.27 ± 0.36
a
 
6.41 ± 0.53
a
 
6.99 ± 0.18
a
 
7.45 ± 0.45
a
 
7.35 ± 0.57
a
 
7.52 ± 0.46
a
 
7.49 ± 0.56
a
 
PI
-
2 (1.00%)
 
2.36 
± 0.72
a
 
3.81 ± 0.19
b
 
5.10 ± 1.01
b
 
5.95 ± 1.48
ab
 
6.27 ± 1.03
ab
 
6.95 ± 0.63
abc
 
7.13 ± 0.52
a
 
PI
-
3 (0.65%)
 
2.50 ± 0.42
a
 
3.34 ± 0.75
b
 
4.28 ± 0.96
bc
 
5.77 ± 0.81
ab
 
6.38 ± 0.82
ab
 
7.14 ± 0.56
ab
 
7.49 ± 0.22
a
 
PI
-
5 (1.00%)
 
2.57 ± 0.20
a
 
3.03 ± 0.43
b
 
3.53 ± 0.37
c
 
4.06
 
± 0.65
b
 
4.88 ± 1.11
b
 
6.08 ± 0.58
c
 
7.33 ± 0.72
a
 
LI
-
4 (2.5%
)
 
2.30 ± 0.52
a
 
3.17 ± 0.24
b
 
5.28 ± 0.38
b
 
5.81 ± 0.58
ab
 
6.17 ± 0.80
ab
 
6.26 ± 0.55
bc
 
6.81 ± 0.21
a
 
Storage at 7
o
C
 
CTR (0%)
 
2.27 ± 0.36
a
 
6.72 ± 0.38
a
 
6.96 ± 0.39
a
 
7.43 ± 0.33
a
 
7.58 ± 0.25
a
 
7.71 ± 0.0
5
a
 
7.90 ± 0.30
a
 
PI
-
2 (1.00%)
 
2.36 ± 0.72
a
 
4.69 ± 0.95
b
 
5.94 ± 0.86
ab
 
6.72 ± 0.54
a
 
6.97 ± 1.19
a
 
7.42 ± 0.37
a
 
7.27 ± 0.40
a
 
PI
-
3 (0.65%)
 
2.50 ± 0.42
a
 
4.30 ± 1.21
bc
 
5.57 ± 0.98
ab
 
6.62 ± 0.49
ab
 
7.15 ± 0.77
a
 
7.22 ± 0.61
ab
 
7.40 ± 0.50
a
 
PI
-
5 (1.00%)
 
2.57 ± 0.20
a
 
3.07 ± 0.62
c
 
4.22 ± 0.44
b
 
4.75 ± 1.19
b
 
5.06 ± 0.84
b
 
6.41 ± 0.15
b
 
7.18 ± 0.73
a
 
LI
-
4 (2.5%
)
 
2.30 ± 0.52
a
 
4.66 ± 0.48
b
 
5.78 ± 1.06
ab
 
5.91 ± 0.58
ab
 
6.67 ± 0.35
a
 
6.95 ± 0.12
ab
 
7.16 ± 0.21
a
 
Storage at 10
o
C
 
CTR (0%)
 
2.27 ± 0.36
a
 
7.32 ± 0.31
a
 
7.82 ± 0.09
a
 
7.4
1 ± 0.02
a
 
7.60 ± 0.50
a
 
7.55 ± 0.30
a
 
7.54 ± 0.22
a
 
PI
-
2 (1.00%)
 
2.36 ± 0.72
a
 
6.04 ± 0.31
b
 
6.95 ± 0.41
a
 
7.19 ± 0.49
ab
 
7.45 ± 0.19
a
 
7.46 ± 0.48
a
 
7.72 ± 0.09
a
 
PI
-
3 (0.65%)
 
2.50 ± 0.42
a
 
5.41 ± 0.68
b
 
6.59 ± 0.65
a
 
6.43 ± 1.23
ab
 
6.94 ± 0.72
ab
 
7.17 ± 0.38
ab
 
7.70 ±
 
0.49
a
 
PI
-
5 (1.00%)
 
2.57 ± 0.20
a
 
3.91 ± 0.13
c
 
4.88 ± 0.65
b
 
5.53 ± 1.02
b
 
6.06 ± 0.67
b
 
6.57 ± 0.64
b
 
7.64 ± 0.49
a
 
LI
-
4 (2.5%
)
 
2.30 ± 0.52
a
 
6.11 ± 0.12
b
 
6.87 ± 0.38
a
 
6.96 ± 0.26
ab
 
7.04 ± 0.21
ab
 
6.91 ± 0.52
ab
 
6.96 ± 0.77
a
 
1
Mean values with 
same letters in th
e same column were
 
not 
significantly different (
P
 

 
0.05).      
 
2
M
eans
 

 
standard deviation
 
of 
n
 
= 6 observations
 
for each reading
, except for control
. 
 
3
The minimum detectability of the methodology was > 10 cells per gram.
 
4
Inhibitors 
in the formulation
 
as in Table 
2.
3
 
 
 
 
 
 
 
 
 
99
 
 
Table 
A
.
7
  
Population
 
of 
L. monocytogenes
1
 
in TSBYE with or without different hop acid extracts at 5 ppm
, 
0.5 or 1% PAPD, 


 
during 6 days of storage at 7
o
C.
 
Treatment
 
 
Populations of 
L. monocytogenes
2
 
(log CFU/mL)
 
Day 1
 
Day 2
 
Day 3
 
Day 4
 
Day 5
 
Day 6
 

-
acid
 
3.68 
+
 
0.18
a
 
4.30 
+
 
0.35
ab
 
4.79 
+
 
0.63
b
 
5.26 
+
 
0.54
c
 
5.69 
+
 
0.64
b
 
6.12 
+
 
0.64
bc
 

-
acid
 
3.80 
+
 
0.73
a
 
3.55 
+
 
0.55
ab
 
3.51 
+
 
0.61
a
 
3.52 
+
 
0.50
ab
 
3.63 
+
 
0.51
a
 
3.76 
+
 
1.02
a
 
acid
-
tetra
 
3.80 
+
 
0.45
a
 
4.14 
+
 
0.47
ab
 
4.28 
+
 
0.22
ab
 
4.96 
+
 
0.84
bc
 
5.33 
+
 
0.71
b
 
5.55 
+
 
0.66
b
 
K
-
tetra
 
4.04 
+
 
0.41
a
 
4.48 
+
 
0.36
ab
 
5.15 
+
 
0.21
b
 
5.80 
+
 
0.31
c
 
6.32 
+
 
0.48
b
 
7.02 
+
 
0.67
bc
 
K
-
hexa
 
4.01 
+
 
0.45
a
 
4.53 
+
 
0.36
ab
 
5.37 
+
 
0.32
b
 
5.96 
+
 
0.41
c
 
6.55 
+
 
0.61
b
 
7.02 
+
 
0.75
bc
 

-
acid/PAPD
 
3.45 
+
 
0.41
a
 
3.27 
+
 
0.46
a
 
3.32 
+
 
0.31
a
 
3.40 
+
 
0.53
a
 
3.09 
+
 
0.44
a
 
3.06 
+
 
0.30
a
 

-
acid/PAPD
 
3.84 
+
 
0.42
a
 
3.40 
+
 
0.40
ab
 
3.36 
+
 
0.39
a
 
3.40 
+
 
0.46
a
 
3.36 
+
 
0.44
a
 
3.35 
+
 
0.49
a
 
acid
-
tetra/PAPD
 
3.71 
+
 
0.60
a
 
3.40 
+
 
0.78
ab
 
3.41 
+
 
0.45
a
 
3.50 
+
 
0.56
ab
 
3.3
9 
+
 
0.58
a
 
3.49 
+
 
0.46
a
 
K
-
tetra/PAPD
 
3.78 
+
 
0.84
a
 
3.56 
+
 
0.55
ab
 
3.60 
+
 
0.52
a
 
3.50 
+
 
0.50
ab
 
3.49 
+
 
0.56
a
 
3.43 
+
 
0.47
a
 
K
-
hexa/PAPD
 
3.79 
+
 
0.68
a
 
3.62 
+
 
0.49
ab
 
3.48 
+
 
0.50
a
 
3.51 
+
 
0.58
ab
 
3.44 
+
 
0.45
a
 
3.41 
+
 
0.35
a
 
0.5%PAPD
 
3.86 
+
 
0.76
a
 
3.59 
+
 
0.51
ab
 
3.55 
+
 
0.
60
a
 
3.55 
+
 
0.61
ab
 
3.61 
+
 
0.61
a
 
3.64 
+
 
0.57
a
 
1%PAPD
 
3.69 
+
 
0.68
a
 
3.48 
+
 
0.47
ab
 
3.44 
+
 
0.51
a
 
3.27 
+
 
0.49
a
 
3.36 
+
 
0.47
a
 
3.33 
+
 
0.39
a
 
TSBYE
 
4.25 
+
 
0.64
a
 
4.74 
+
 
0.41
b
 
5.36
 
+
 
0.30
b
 
6.14 
+
 
0.46
c
 
6.67
 
+
 
0.75
b
 
7.26
 
+
 
0.99
c
 
1
Listeria
 
inoculated with 
3.8
4
 

 
0.28 l
og CFU/mL
.
  
 
2
Means 

 
standard deviation of 
n
 
= 6 observations
 
for each reading
.
 
a
-
c
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05).
 
 
100
 
 
Table A.8
  
Population
 
of 
L. monocytogenes
1,2
 
on 
vacuum
-
packaged deli
-
style tu
rkey meat with various inhibitors during 60 days of 
storage at 4 and 
7
o
C.
 
 
Treatment
 
Population of 
Listeria monocytogenes
 
(log CFU/g
) on storage day
 
Day 0
 
Day 7
 
Day 15
 
Day 30
 
Day 45
 
Day 60
 
Storage at 4
 
o
C
 
 
 
 
 
 
 
CTR
 
 
2.39
 

 
0.36 
a
 
2.96
 

 
0.11 
a
 
3.73
 

 
0
.37
 
b
 
5.18
 

 
0.13
 
b
 
6.52
 

 
0.38
 
b
 
7.54
 

 
0.13
 
b
 
PLSD
 
 
2.48 

 
0.52
 
a
 
 
2.33 

 
0.06
 
a
 
 
2.37 

 
0.31
 
a
 
 
2.37 

 
0.34
 
a
 
 
2.36 

 
0.37
 
a
 
 
2.39 

 
0.36
 
a 
 
PAPD
 
 
2.56 

 
0.37
 
a
 
 
2.55 

 
0.29
 
a
 
 
2.37 

 
0.15
 
a
 
 
2.32 

 
0.32
 
a
 
 
2.54 

 
0.60
 
a
 
2.73
 

 
0.51
 
a
 

-
acid
 
 
2.28 

 
0.55
 
a
 
 
2.65 

 
0.18
 
a
 
 
3.56 

 
0.43
 
b
 
 
4.57 

 
0.14
 
b
 
 
6.40 

 
0.42
 
b
 
 
7.02 

 
0.48
 
b
 

-
acid/
PAPD
 
 
2.46 

 
0.17
 
a
 
 
2.42 

 
0.31
 
a
 
 
2.39 

 
0.26
 
a
 
 
2.54 

 
0.47
 
a
 
 
2.54 

 
0.34
 
a
 
 
2.61 

 
0.60
 
a
 

-
acid
 
 
2.59 

 
0.27
 
a
 
 
2.72 

 
0.12
 
a
 
 
3.40 

 
0.22
 
b
 
 
4.50 

 
0.43
 
b
 
 
6.
42 

 
0.43
 
b
 
 
7.14 

 
0.18
 
b
 

-
acid/
PAPD
 
 
2.49 

 
0.29
 
a
 
 
2.43 

 
0.38
 
a
 
 
2.24 

 
0.14
 
a
 
 
2.27 

 
0.47
 
a
 
 
2.37 

 
0.38
 
a
 
 
2.39 

 
0.44
 
a
 
Storage at 7
 
o
C
 
 
 
 
 
 
 
CTR
 
 
2.39
 

 
0.36 
x
 
3.60
 

 
0.51 
y
 
5.73
 

 
0.33
 
y
 
7.65
 

 
0.24
 
y
 
8.48
 

 
0.19
 
y
 
8.29
 

 
0.10
 
z
 
PLSD
 
 
2.48 

 
0.52
 
x
 
 
2.51 

 
0.36 
xy
 
 
2.68 

 
0.50
 
x
 
 
3.60 

 
0.63
 
x
 
 
4.71 

 
0.99
 
x
 
 
5.64 

 
1.04
 
y
 
PAPD
 
 
2.56 

 
0.37
 
x
 
 
2.55 

 
0.32 
xy
 
 
2.70 

 
0.25
 
x
 
 
3.37 

 
0.50
 
x
 
 
3.99 

 
0.77
 
x
 
 
4.80 

 
0.39
 
xy
 

-
acid
 
 
2.28 

 
0.55
 
x
 
 
3.54 

 
0.49 
y
 
 
5.65 

 
0.37
 
y
 
 
7.54 

 
0.25
 
y
 
 
8.26 

 
0.16
 
y
 
 
8.03 

 
0.11
 
z
 

-
acid/
PAPD
 
 
2.46 

 
0.17
 
x
 
 
2.58 

 
0.36 
xy
 
 
2.38 

 
0.28
 
x
 
 
3.25 

 
0.54
 
x
 
 
3.69 

 
0.77
 
x
 
 
4.37 

 
0.59
 
xy
 

-
acid
 
 
2.59 

 
0.27
 
x
 
 
3.59 

 
0.55 
y
 
 
5.54 

 
0.48
 
y
 
 
7.69 

 
0.01
 
y
 
 
8.30 

 
0.33
 
y
 
 
8.18 

 
0.35
 
z
 

-
acid/
PAPD
 
 
2.49 

 
0.29
 
x
 
 
2.38 

 
0.11 
x
 
 
2.75 

 
0.02
 
x
 
 
3.30 

 
0.19
 
x
 
 
4.02 

 
0.41
 
x
 
 
4.13 

 
0.78
 
x
 
1
Means 

 
standard deviation of 
n
 
= 6 observations
 
for each reading
.
 
2
The minimum detectability of the methodology was > 10 cells per gram. 
 
a
-
c
, x
-
z
Mean values with 
same letters in 
the same column were
 
not 
significantly different (
P
 

 
0.05).
 
101
 
 
Table A.9
  
Population of 
L. monocytogenes
1,2
 
at 4 and 
7
o
C 
on aerobic
-
packaged deli
-
style turkey meat with various inhibitors and 
sliced after 30 days of storage whole sticks
.
 
 
Treatment
 
Populati
on of 
Listeria monocytogenes
 
(log CFU/g
) on storage day
 
Day 0
 
Day 2
 
Day 4
 
Day 6
 
Day 8
 
Day 10
 
Storage at 4
 
o
C
 
 
 
 
 
 
 
CTR
 
2.43
 

 
0.41
 
a
 
 
2.39
 

 
0.36 
a
 
 
2.47
 

 
0.21
 
a
 
 
2.68
 

 
0.24
 
a
 
 
2.63 

 
0.57
 
a
 
 
2.97 

 
0.43
 
a
 
PLSD
 
 
2.42 

 
0.43
 
a
 
 
1.97 

 
0.85
 
a
 
 
2.28 

 
0.50
 
a
 
 
2.36 

 
0.10
 
a
 
 
1.99 

 
0.85
 
a
 
 
2.24 

 
0.47
 
a
 
PAPD
 
 
2.45 

 
0.30
 
a
 
 
2.35 

 
0.37
 
a
 
 
2.08 

 
0.43
 
a
 
 
2.13 

 
0.38
 
a
 
 
2.33 

 
0.3
1
 
a
 
 
1.87 

 
0.81
 
a
 

-
acid
 
 
2.20 

 
0.35
 
a
 
 
2.35 

 
0.56
 
a
 
 
1.91 

 
0.81
 
a
 
 
2.43 

 
0.10
 
a
 
 
2.63 

 
0.54
 
a
 
 
3.05 

 
0.16
 
a
 

-
acid/
PAPD
 
 
2.39 

 
0.45
 
a
 
 
2.38 

 
0.33
 
a
 
 
2.16 

 
0.41
 
a
 
 
1.83 

 
0.76
 
a
 
 
1.99 

 
0.85
 
a
 
 
2.38
 

 
0.17
 
a
 

-
acid
 
 
2.18 

 
0.60
 
a
 
 
2.52 

 
0.31
 
a
 
 
2.52 

 
0.45
 
a
 
 
2.
78 

 
0.42
 
a
 
 
2.84 

 
0.35
 
a
 
 
3.08 

 
0.07
 
a
 

-
acid/
PAPD
 
 
2.32 

 
0.58
 
a
 
 
2.10 

 
0.35
 
a
 
 
2.13 

 
0.38
 
a
 
 
2.36 

 
0.10
 
a
 
 
1.77 

 
0.68
 
a
 
 
1.83
 

 
0.72
 
a
 
Storage at 7
 
o
C
 
 
 
 
 
 
 
CTR
 
2.43
 

 
0.41
 
x
 
 
2.76
 

 
0.27
 
x
 
2.87
 

 
0.51
 
x
 
3.55
 

 
0.41
 
y
 
 
4.09
 

 
0.12
 
y
 
4.44
 

 
0.21
 
z
 
PLSD
 
 
2.42 

 
0.43
 
x
 
 
2.41 

 
0.37
 
x
 
2.21
 

 
0.45
 
x
 
 
2.12 

 
0.39
 
x
 
 
2.50 

 
0.44
 
x
 
 
2.59 

 
0.36
 
xy
 
PAPD
 
 
2.45 

 
0.30
 
x
 
 
2.00 

 
0.87
 
x
 
 
2.35 

 
0.16
 
x
 
 
2.35 

 
0.15
 
xy
 
 
2.12 

 
0.39
 
x
 
 
2.32 

 
0.28
 
x
 

-
acid
 
 
2.20 

 
0.35
 
x
 
 
2.47 

 
0.66
 
x
 
 
2.80 

 
0.54
 
x
 
 
3.21 

 
0.37
 
xy
 
 
3.54 

 
0.16
 
y
 
 
4.10 

 
0.57
 
yz
 

-
acid/
PAPD
 
 
2.39 

 
0.45
 
x
 
 
2.43 

 
0.15
 
x
 
 
2.19
 

 
0.43
 
x
 
 
2.22 

 
0.24
 
x
 
 
2.32
 

 
0.28
 
x
 
 
1.90 

 
0.78
 
x
 

-
acid
 
 
2.18 

 
0.60
 
x
 
 
2.25 

 
0.99
 
x
 
 
2.90 

 
0.34
 
x
 
 
3.53 

 
0.20
 
y
 
 
4.04 

 
0.16
 
y
 
 
4.17 

 
0.08
 
z
 

-
acid/
PAPD
 
 
2
.32 

 
0.58
 
x
 
 
2.08 

 
0.43
 
x
 
 
2.20 

 
0.46
 
x
 
 
2.08 

 
0.94
 
x
 
 
2.13 

 
0.23
 
x
 
 
2.09 

 
0.95
 
x
 
1
Means 

 
standard deviation of 
n
 
= 6 observations
 
for each reading
.
 
2
The minimum detectability of the methodology was > 10 cells per gram. 
 
a
-
c
, x
-
z
Mean values with 
sa
me letters in the same column were
 
not 
significantly different (
P
 

 
0.05).
 
 
 
 
102
 
 
Table A.10
 
 
Population of 
L. monocytogenes
1,2
 
at 4 and 
7
o
C 
on aerobic
-
packaged deli
-
style turkey meat with various inhibitors and 
sliced after 60 days of storage whole sticks
.
 
 
T
reatment
 
Population of 
Listeria monocytogenes
 
(log CFU/g
) on storage day
 
Day 0
 
Day 2
 
Day 4
 
Day 6
 
Day 8
 
Day 10
 
Storage at 4
 
o
C
 
 
 
 
 
 
 
CTR
 
2.76
 

 
0.23
 
a
 
2.73
 

 
0.24
 
a
 
2.84
 

 
0.39
 
a
 
2.98
 

 
0.05
 
a
 
3.06
 

 
0.04
 
a
 
3.21
 

 
0.06
 
a
 
PLSD
 
2.67
 

 
0.45
 
a
 
2.70
 

 
0.33
 
a
 
2.66
 

 
0.26
 
a
 
2.59
 

 
0.27
 
a
 
2.67
 

 
0.28
 
a
 
2.66
 

 
0.22
 
a
 
PAPD
 
2.78
 

 
0.35
 
a
 
2.69
 

 
0.31
 
a
 
2.61
 

 
0.34
 
a
 
2.62
 

 
0.31
 
a
 
2.66
 

 
0.26
 
a
 
2.53
 

 
0.13
 
a
 

-
acid
 
2.73
 

 
0.34
 
a
 
2.72
 

 
0.26
 
a
 
2.59
 

 
0.36
 
a
 
2.56
 

 
0.28
 
a
 
2.65
 

 
0.37
 
a
 
2.74
 

 
0.24
 
a
 

-
acid/
PAPD
 
2.78
 

 
0.11
 
a
 
2.50
 

 
0.35
 
a
 
2.54
 

 
0.34
 
a
 
2.63
 

 
0.38
 
a
 
2.56
 

 
0.28
 
a
 
2.67
 

 
0.36
 
a
 

-
acid
 
2.66
 

 
0.26
 
a
 
2.59
 

 
0.41
 
a
 
2.66
 

 
0.55
 
a
 
2.83
 

 
0.63
 
a
 
3.06
 

 
0.65
 
a
 
3.27
 

 
0.86
 
a
 

-
acid/
PAPD
 
 
2.78 

 
0.19
 
a
 
2.61
 

 
0.32
 
a
 
2.47
 

 
0.29
 
a
 
2.45
 

 
0.18
 
a
 
2.59
 

 
0.26
 
a
 
2.56
 

 
0.17
 
a
 
Storage at 7
 
o
C
 
 
 
 
 
 
 
CTR
 
 
2.76 

 
0.23
 
x
 
2.92
 

 
0.17
 
x
 
3.00
 

 
0.03
 
x
 
3.29
 

 
0.13
 
x
 
3.65
 

 
0.01
 
y
 
4.08
 

 
0.16
 
y
 
PLSD
 
 
2.67 

 
0.45
 
x
 
2.63
 

 
0.16
 
x
 
2.68
 

 
0.19
 
x
 
2.73
 

 
0.21
 
x
 
2.76
 

 
0.23
 
x
 
2.84
 

 
0.28
 
x
 
PAPD
 
2.78
 

 
0.35
 
x
 
2.73
 

 
0.33
 
x
 
2.68
 

 
0.24
 
x
 
2.64
 

 
0.19
 
x
 
2.68
 

 
0.24
 
x
 
2.75
 

 
0.25
 
x
 

-
acid
 
 
2.73 

 
0.34
 
x
 
2.76
 

 
0.26
 
x
 
2.68
 

 
0.07
 
x
 
2.79
 

 
0.20
 
x
 
2.84
 

 
0.10
 
x
 
3.05
 

 
0.09
 
x
 

-
acid/
PAPD
 
2.78
 

 
0.11
 
x
 
2.72
 

 
0.32
 
x
 
2.69
 

 
0.24
 
x
 
2.71
 

 
0.18
 
x
 
2.72
 

 
0.23
 
x
 
2.80
 

 
0.33
 
x
 

-
acid
 
2.66
 

 
0.26
 
x
 
2.73
 

 
0.38
 
x
 
3.00
 

 
0.80
 
x
 
3.20
 

 
1.04
 
x
 
3.50
 

 
1.23
 
y
 
3.87
 

 
1.32
 
y
 

-
acid/
PAPD
 
 
2.78 

 
0.19
 
x
 
2.73
 

 
0.26
 
x
 
2.58
 

 
0.09
 
x
 
2.70
 

 
0.28
 
x
 
2.63
 

 
0.08
 
x
 
2.67
 

 
0.18
 
x
 
1
Means 

 
standard deviation of 
n
 
= 6 observations
 
for each reading
.
 
2
The minimum detectability of the methodology was > 10 cells per gram. 
 
a
-
b
, x
-
y
Mean values with 
same letters in the same column were
 
not 
significantly differe
nt (
P
 

 
0.05).
 
 
 
 
103
 
 
Table A.11
  
Population
 
of 
L. monocytogenes
1,2
 
(log CFU/mL)
 
in skim milk
 
with or without diffe
rent hop acid extracts at 5 ppm
 
or 
0.5
% PAPD during 6 days of storage at 7
o
C.
 
 
 
Treatment
 
Storage time (day)
 
1
 
2
 
3
 
4
 
5
 
6
 

-
acid
 
 
4.19 ± 0.27
a
 
4.31 ± 0.23
a
 
4.41 ± 0.50
a
 
4.71 ± 0.53
ab
 
4.97 ± 0.76
ab
 
5.13 ± 0.80
abc
 

-
acid
 
4.20 ± 0.21
a
 
4.43 ± 0.38
a
 
4.59 ± 0.44
a
 
4.90 ± 0.40
ab
 
5.06 ± 0.71
ab
 
5.13 ± 0.73
abc
 
acid
-
tetra
 
4.20 ± 0.23
a
 
4.28 ± 0.43
a
 
4.21 ± 0.39
a
 
4.37 ± 0.33
ab
 
4.53 ± 0.49
ab
 
4.67 ± 0.60
ab
 
K
-
tetra
 
4.13 ± 0.26
a
 
4.23 ± 0.56
a
 
4.25 ± 0.21
a
 
4.22 ± 0.30
ab
 
4.27 ± 0.45
ab
 
4.51 ± 0.43
ab
 
K
-
hexa
 
4.20 ± 0.22
a
 
4.38 ± 0.44
a
 
4.34 ± 0.53
a
 
4.63 ± 0.51
ab
 
4.84 ± 0.62
ab
 
4.98 ± 0.63
abc
 

-
acid/PAPD
 
4.14 ± 0.20
a
 
4.18 ± 0.66
a
 
3.96 ± 0.43
a
 
3.95 ± 
0.32
a
 
3.98 ± 0.42
a
 
3.93 ± 0.37
a
 

-
acid/PAPD
 
4.08 ± 0.29
a
 
4.16 ± 0.53
a
 
4.11 ± 0.33
a
 
3.98 ± 0.35
a
 
3.95 ± 0.31
a
 
3.92 ± 0.24
a
 
acid
-
tetra/PAPD
 
4.10 ± 0.29
a
 
4.13 ± 0.62
a
 
3.81 ± 0.51
a
 
3.99 ± 0.29
a
 
4.00 ± 0.36
a
 
4.03 ± 0.35
a
 
K
-
tetra/PAPD
 
4.03 ± 0.38
a
 
4.23 ± 0.53
a
 
3.86 ± 0.37
a
 
4.00 ± 0.35
a
 
3.92 ± 0.48
a
 
3.97 ± 0.33
a
 
K
-
hexa/PAPD
 
4.03 ± 0.37
a
 
4.23 ± 0.57
a
 
3.94 ± 0.25
a
 
4.00 ± 0.40
a
 
3.98 ± 0.34
a
 
3.92 ± 0.30
a
 
0.5%PAPD
 
4.11 ± 0.20
a
 
4.15 ± 0.53
a
 
3.89 ± 0.38
a
 
3.93 ± 0.33
a
 
3.96 ± 0.35
a
 
3.98 ± 0.30
a
 
Skim
 
milk
 
4.34 ± 0.26
a
 
4.55 ± 0.45
a
 
4.74 ± 0.42
a
 
5.16 ± 0.65
ab
 
5.56 ± 0.66
b
 
5.91 ± 0.52
bc
 
TSBYE
 
4.29 ± 0.24
a
 
 
4.39 ± 0.34
a
 
4.84
 
± 0.62
a
 
5.26 ± 0.65
b
 
5.63 ± 0.49
b
 
6.10
 
 
± 0.32
c
 
1
Means 

 
standard deviation of 
n
 
= 6 observations
 
for each reading
.
 
2
The minimum detectability of th
e methodology was > 10 cells per gram. 
 
a
-
c
 
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05).
 
 
 
104
 
 
Table A.12
  
Population
 
of 
L. monocytogenes
1,2
 
(log CFU/mL)
 
in 2% milk
 
with or without diffe
rent hop acid extracts at
 
5 ppm
 
or 0.5
% 
PAPD during 6 days of storage at 7
o
C.
 
 
 
Treatment
 
Storage time (day)
 
1
 
2
 
3
 
4
 
5
 
6
 

-
acid
 
 
4.16 ± 0.22
a
 
 
4.35 ± 0.30
a
 
 
4.52 ± 0.47
a
 
 
4.76 ± 0.61
a
 
 
5.07 ± 0.86
a
 
 
5.24 ± 0.90
ab
 

-
acid
 
4.17 ± 0.21
a
 
4.33 ± 0.36
a
 
4.56 ± 0.48
a
 
4.89 ± 0.74
a
 
5.12 ± 0.86
a
 
5.29 ± 0.93
ab
 
acid
-
tetra
 
4.11 ± 0.34
a
 
4.25 ± 0.27
a
 
4.34 ± 0.40
a
 
4.50 ± 0.45
a
 
4.80 ± 0.81
a
 
4.95 ± 1.03
ab
 
K
-
tetra
 
4.14 ± 0.27
a
 
4.24 ± 0.35
a
 
4.33 ± 0.31
a
 
4.46 ± 0.45
a
 
4.72 ± 0.82
a
 
4.94 ± 0.85
ab
 
K
-
hexa
 
4.20 ± 0.22
a
 
4.24 ± 0.42
a
 
4.41 ± 0.43
a
 
4.66 ± 0.63
a
 
4.51 ± 1.17
a
 
5.11 ± 0.81
ab
 

-
acid/PAPD
 
4.00 ± 0.34
a
 
4.16 ± 0.30
a
 
3.98 ± 0.42
a
 
4.01 ± 0.30
a
 
3.
86 ± 0.41
a
 
3.85 ± 0.42
a
 

-
acid/PAPD
 
4.14 ± 0.21
a
 
4.04 ± 0.36
a
 
4.04 ± 0.39
a
 
3.96 ± 0.31
a
 
3.95 ± 0.37
a
 
3.95 ± 0.36
a
 
acid
-
tetra/PAPD
 
4.04 ± 0.36
a
 
4.06 ± 0.39
a
 
4.02 ± 0.42
a
 
4.04 ± 0.37
a
 
3.98 ± 0.29
a
 
3.94 ± 0.30
a
 
K
-
tetra/PAPD
 
4.06 ± 0.31
a
 
4.04 ± 0.42
a
 
4.02 ±
 
0.37
a
 
3.94 ± 0.36
a
 
3.95 ± 0.38
a
 
3.88 ± 0.38
a
 
K
-
hexa/PAPD
 
4.09 ± 0.23
a
 
3.95 ± 0.41
a
 
3.94 ± 0.49
a
 
3.93 ± 0.30
a
 
3.96 ± 0.40
a
 
3.96 ± 0.40
a
 
0.5%PAPD
 
4.00 ± 0.31
a
 
4.10 ± 0.23
a
 
4.06 ± 0.30
a
 
3.99 ± 0.42
a
 
3.98 ± 0.50
a
 
3.88 ± 0.29
a
 
2% Milk
 
4.29 ± 0.23
a
 
4.35 ± 0.
29
a
 
4.63 ± 0.39
a
 
5.13 ± 0.59
a
 
5.63 ± 0.49
a
 
5.76 ± 0.68
ab
 
TSBYE
 
4.29 ± 0.24
a
 
 
4.39 ± 0.34
a
 
4.84
 
± 0.62
a
 
5.26 ± 0.65
a
 
5.63 ± 0.49
a
 
6.10
 
 
± 0.32
b
 
1
Means 

 
standard deviation of 
n
 
= 6 observations
 
for each
 
re
a
ding
.
 
2
The minimum detectability of the methodology was > 10 cells per gram. 
 
a
-
c
 
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05).
 
 
 
 
105
 
 
Table A.13
  
Population of 
L. monocytogenes
1,2
 
(log CFU/g)
 
in 
turkey slurries
 
contain
ing 

-
acid
 

-
acid
 
at 0 to 1000 ppm 
during 12 
days of storage at 7
o
C.
 
Treatment
 
Number of 
L. monocytogenes 
(log CFU/g)
 
Day 0
 
Day 3
 
Day 6
 
Day 9
 
Day 12
 

-
acid 5 ppm
 
2.43 
+
 
0.43
 
a, A
 
3.44 
+
 
0.26 
d, AB
 
4.49
 
+
 
0.34 
d, 
B
 
5.72
 
+
 
0.49 
b, C
 
6.71
 
+
 
0.52 
b, C
 

-
aci
d 25 ppm
 
2.37 
+
 
0.45 
a, A
 
3.10
 
+
 
0.58 
bcd, AB
 
4.21
 
+
 
0.34 
d, BC
 
5.35
 
+
 
0.63 
b, CD
 
6.14
 
+
 
0.62 
b, D
 

-
acid 50 ppm
 
2.45 
+
 
0.43 
a, A
 
2.46
 
+
 
0.45 
abcd, A
 
3.44
 
+
 
0.28 
cd, AB
 
4.41
 
+
 
0.48 
b, BC
 
5.19
 
+
 
0.63 
b, C
 

-
acid 100 ppm
 
2.37 
+
 
0.33 
a, A
 
2.33
 
+
 
0.43 
abcd, 
A
 
2.44
 
+
 
0.52 
abc, A
 
2.64
 
+
 
0.48 
a, A
 
3.18
 
+
 
0.70 
a, A
 

-
acid 500 ppm
 
2.27 
+
 
0.30 
a, A
 
2.18
 
+
 
0.37 
abc, A
 
2.20
 
+
 
0.48 
ab, A
 
2.18
 
+
 
0.58
 
a, A
 
2.10
 
+
 
0.47 
a, A
 

-
acid 1000 ppm
 
2.23 
+
 
0.44 
a, A
 
1.92
 
+
 
0.42 
a, A
 
1.85
 
+
 
0.39 
a, A
 
1.77
 
+
 
0.46 
a, A
 
1.83
 
+
 
0.50 
a, A
 

-
acid 5 ppm
 
2.41 
+
 
0.44 
a, A
 
3.36
 
+
 
0.31 
d, A
 
4.57
 
+
 
0.38 
d, B
 
5.75
 
+
 
0.43 
b, C
 
6.54
 
+
 
0.52 
b, C
 

-
acid 25 ppm
 
2.43 
+
 
0.46 
a, A
 
3.25
 
+
 
0.32 
cd, AB
 
4.41
 
+
 
0.32 
d, BC
 
5.48
 
+
 
0.59 
b, CD
 
6.09
 
+
 
0.60 
b, D
 

-
acid 50 ppm
 
2.42 
+
 
0.44 
a, A
 
2.90
 
+
 
0.37 
abcd
, A
 
4.28
 
+
 
0.29 
d, B
 
5.33
 
+
 
0.45
 
b, BC
 
5.83
 
+
 
0.70 
b, C
 

-
acid 100 ppm
 
2.36 
+
 
0.46 
a, A
 
2.62
 
+
 
0.36 
abcd, A
 
3.38
 
+
 
0.27 
bcd, A
 
4.84
 
+
 
0.49 
b, 
B
 
5.54
 
+
 
0.73
 
b, B
 

-
acid 500 ppm
 
 
2.40 
+
 
0.43 
a, A
 
2.37
 
+
 
0.49 
abcd, A
 
2.24
 
+
 
0.63 
ab, A
 
2.29
 
+
 
0.66 
a, A
 
2.10
 
+
 
0.60 
a, A
 

-
acid 1000 ppm
 
2.35 
+
 
0.42 
a, A
 
1.97
 
+
 
0.43 
ab, A
 
1.79
 
+
 
0.45 
a, A
 
1.54
 
+
 
0.53 
a, A
 
1.80
 
+
 
0.71 
a, A
 
Control 0 ppm
 
2.47 
+
 
0.36 
a, A
 
3.48
 
+
 
0.23 
d, B
 
4.57
 
+
 
0.38 
d, C
 
5.83
 
+
 
0.41 
b, D
 
 
6.88
 
+
 
0.61 
b, E
 
1
Means 

 
standard deviation of 
n
 
= 6 ob
servations
 
for each reading
.
 
2
The minimum detectability of the methodology was > 10 cells per gram. 
 
a
-
d 
Mean values with 
same letters in the same column were
 
not 
significantly different (
P
 

 
0.05).
 
A
-
E
Mean values with 
same letters in the same row were
 
not
 
significantly different (
P
 

 
0.05).
 
106
 
 
APPENDIX B
 
 
Calculation of combination index (CI)
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
107
 
 
Calculation of combination index (CI)
 
In Chapter 3, the combination index (CI) was calculated to determine the synergistic 
effect of hop/PAPD combin
ation in 
Listeria 
inhibition.  CI was the result of sum of log
-
reduction 
(comparing to initial inoculation) from individual treatment dividing by log
-
reduction from 
combination treatment.  The results were interpreted as synergistic (CI < 1), additive (CI 
= 1), 
and antagonistic (CI > 1).  However, the results showed in Table 3.2 are calculated based on 
mean values of log reduction, which might lack of degree of uncertainty.  To be more realistic, 
the calculation of CI should use all data from the experiment
 
and the CI should be presented in 
range of value (Table B.1).  
 
Table
 
B.1
  
Interpretation 
possible 
effects of 








 
Treatment
 
Log
-
reduc
tion 
from hop 
acid or PAPD 
alone
 
Log
-
reduction 
from 
hop/PAPD
 
Sum of log
-
reduction 
from hop 
acid alone 
and PAPD 
alone
 
Possib
le CI
 
Interpretation
 

-
acid
 
3.67 
-
 
4.41
 
4.57 
-
 
4.95
 
5.04 
-
 
6.36
 
1.02 
-
 
1.39
 
A
N
 

-
acid
 
1.86 
-
 
2.25
 
4.57 
-
 
4.95
 
3.23 
-
 
4.20
 
0.65 
-
 
0.92
 
SY
 
Acid
-
tetra
 
1.67 
-
 
2.77
 
4.57 
-
 
4.95
 
3.04 
-
 
4.72
 
0.61 
-
 
1.03
 
AN/SY/AD
 
K
-
tetra
 
1.79 
-
 
2.23
 
3.47 
-
 
4.95
 
3.16 
-
 
4.18
 
0.64 
-
 
1.20
 
AN/SY/AD
 
K
-
h
exa
 
1.43 
-
 
2.07
 
3.04 
-
 
4.95
 
2.80 
-
 
4.02
 
0.57 
-
 
1.38
 
AN/SY/AD
 
PAPD
 
1.37 
-
 
1.95
 
 
 
 
 
 
 
-
 
1
 
AN: Antagonistic, SY: Synergistic, AD: Additive. 
 
 
 
 
 
108
 
 
Example of 
CI calculation
:
 
1.
 
CI from mean value of log reduction.
 
Given: mean of l
og r
eduction 
from 

-
acid is 3.
94
.  
 
          
 
mean of l
og r
eduction from 
PAPD is 1.59
.  
 
mean of l
og r
eduction from 

-
acid/PAPD is 4
.74.
 
 
Therefore, 
sum of log reduction from 

-
acid alone and PAPD alone is 
5.53
 
(
from 3.94
+1
.59).  
 
      
CI is 1.17 (from 5.53/4.74).
 
 
2.
 
CI from all values o
f log reduction.
 
Given: l
og reduction of from 

-
acid is 3.67 to 4.41 units
 
(data from experiment)
.  
 
          
 
l
og reduction of from 
PAPD is 1.37 to 1.95 units
 
(data from experiment)
.  
 

-
acid/PAPD is 4.57 to 4.95 units
 
(data from exper
iment)
. 
 
Therefore, 
sum of log reduction from 

-
acid alone and PAPD alone is 5.04 (
from 
3.67+1.37) to 6.36 
(
from 
4.41+1.95).
  
 
      
Possible CI is 1.02 (from 5.04/4.95) to 1.39 (from 6.36/4.57).
 
 
 
 
 
 
109
 
 
 
 
 
 
 
 
 
 
 
 
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