Comparative Evaluation of the Aerobic Procedure for Lactic Acid - 3M

20 Pages·2011·0.39 MB·English

June 24, 2011
A Comparative Evaluation of the Aerobic Procedure for Lactic
Acid Bacteria with 3M™ Petrifilm™ Aerobic Count Plates with
Two Reference Methods for the Enumeration of Lactic Acid
Bacteria in Food and Environmental Surfaces
Kiel Fisher, Erin Crowley, Patrick Bird, Megan Boyle, Katherine Goetz, Q Laboratories, Inc.
1400 Harrison Avenue
M. Joseph Benzinger Jr., Mark Juenger, Travis Huffman, James Agin, David Goins
Cincinnati, OH 45214
A validation study of the Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M™ Petrifilm™ Aerobic Count (AC) Plates for the
enumeration and detection of lactic acid bacteria was conducted at Q Laboratories, Inc., Cincinnati, Ohio. The PLAB counts were
compared to the counts obtained by the agars used in two reference methods: The Compendium of Methods for the Microbiological
Examination of Foods, Chapter 19 and ISO 15214: Microbiology of food and animal feeding stuffs—Horizontal method for the
enumeration of mesophilic lactic acid bacteria—Colony-count technique. The Compendium reference agar, All Purpose Tween agar
plus 2% sucrose and bromocresol purple (APTS) and the ISO reference agar, deMan, Rogasa, Sharpe plus bromocresol purple (MRS)
were used in the in the analysis of both food and environmental swab samples. Four food matrices and one environmental surface
were used in the comparative evaluation: salad dressings, ready to eat (RTE) salads, RTE meats, raw meats and stainless steel
surfaces. For those matrices that were inoculated, various brands of each matrix were spiked with Lactobacillus fermentum ATCC
14931 or Weissella viridescens ATCC 12706 at two target recovery levels on 3M Petrifilm AC Plates: a low level of 1–15 CFU/ plate and
a medium level of 15–75 CFU/ plate. Natural lactic acid bacteria contamination was recovered in five brands of RTE meat and in all
brands of raw meat. A paired t-test, comparing the PLAB procedure and the reference methods at varying time points, was conducted
at the 95% confidence level. Statistically comparable mean log values were determined for salad dressings, RTE salads, RTE meats,
and stainless steel surfaces. The PLAB procedure demonstrated reliability as a rapid and selective quantitative method for the
enumeration of lactic acid producing bacteria.
This report presents the analytical results for comparison of the Aerobic Procedure for Lactic Acid Bacteria (PLAB) with
3M™ Petrifilm™ Aerobic Count (AC) Plates to agars used in The Compendium of Methods for the Microbiological Examination
of Foods, Chapter 19 and ISO 15214: Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of
mesophilic lactic acid bacteria—Colony-count technique. All analyses were conducted at Q Laboratories, Inc. (Cincinnati, OH).
3M Petrifilm AC Plates and Aerobic Lactic Bacteria Broth were provided by 3M (Minneapolis, MN).
Q Laboratories, Inc. is an independent company and is not affiliated with, nor a subsidiary of 3M. Q Laboratories, Inc. was contracted to perform an independent study by and
for 3M and the data generated by Q Laboratories, Inc. during the execution of the study does not represent an endorsement of any product(s) by Q Laboratories, Inc.
Page 1
Materials and Methods
The methodology for this study was followed as outlined in the protocol: Comparison of a 3M™ Petrifilm™ Aerobic Count Plate
Method with Three Methods for the Enumeration of Lactic Acid Bacteria in Food and Environmental Samples, provided by 3M.
The study objective was to evaluate the performance of the Aerobic Procedure for Lactic Acid Bacteria with 3M™ Petrifilm™
Aerobic Count (AC) Plates to the ISO and Compendium Reference Methods for detecting and enumerating lactic acid bacteria.
The following matrices were analyzed: salad dressings, ready to eat (RTE) salads, RTE meats, raw meats, and stainless steel
surfaces. A detailed list of the brands used in the food analysis is presented in Table A.
Table A. Matrix Details
Identification Brand
Salad Dressings RTE Salads
Caesar 1 Cardini’s Caesar Dressing Mustard Potato 1 Meijer Markets Mustard Potato Salad
Caesar 2 Newman’s Own Caesar Dressing Mustard Potato 2 Grandma’s Mustard Potato Salad
LoFat Caesar 1 Cardini’s Light Caesar Dressing Mustard Potato 3 Kroger Mustard Potato Salad
LoFat Caesar 2 Ken’s Steak House Light Caesar Dressing American Potato 1 Grandma’s Potato Salad with Egg
Ranch 1 Kroger Creamy Ranch Dressing American Potato 2 Meijer Markets American Potato Salad
Ranch 2 Ken’s Steak House Ranch Dressing American Potato 3 Kroger American Potato Salad
LoFat Ranch 1 Kroger Lite Creamy Ranch Dressing Macaroni 1 Kroger Macaroni Salad
LoFat Ranch 2 Kraft Light Ranch Reduced Fat Dressing Macaroni 2 Meijer Macaroni Salad
Catalina 1 Kraft Classic Catalina Dressing Plus Dip Macaroni 3 Grandmas’ Macaroni Salad
Catalina 2 Kroger California French Dressing Bean 1 Kroger Baked Beans
Blue Cheese 1 Kraft Roka Brand Blue Cheese Dressing Bean 2 Gordon Food Services Baked Beans
Blue Cheese 2 Kroger Chunky Blue Cheese Dressing Bean 3 Meijer Baked Beans
Blue Cheese 3 Ken’s Steak House Chunky Blue Cheese Dressing Coleslaw 1 Meijer Cole Slaw
Zesty Italian 1 Kroger Zesty Italian Dressing Coleslaw 2 Kroger Cole Slaw
Zesty Italian 2 Kraft Zesty Italian Dressing Coleslaw 3 Grandma’s Cole Slaw
RTE Meats Raw Meats
O/R Turkey 1 Sara Lee Fresh Ideas Oven Roasted Turkey Breast Ground Turkey 1 Honeysuckle White Ground Turkey 93/7
O/R Turkey 2 Kroger Deli Thin Sliced Oven Roasted Turkey Breast Ground Turkey 2 Kroger Ground Turkey 85/15
Roast Beef 1 Kroger Deli Thin Sliced Roast Beef Chicken 1 Tyson All Natural Chicken Breast Tenderloins 85/15
Roast Beef 2 Private Selection Homestyle Slow Roasted Roast Beef Chicken 2 Perdue Fresh Ground Chicken
O/R Chicken 1 Private Selection Homestyle Slow Roasted Chicken Breast Ground Beef 1 Kroger Ground Sirloin 90/10
O/R Chicken 2 Sara Lee Fresh Ideas Oven Roasted Chicken Breast Ground Beef 2 Laura’s Lean Ground Beef 92/8
Smoked Ham 1 Private Selection Smokehouse Hickory Ham Steak 1 Kroger Beef Top Sirloin
Smoked Ham 2 Kroger Deli Think Sliced Smoked Ham Steak 2 Kroger Beef Bottom Round Steak Choice
Culture Preparation and Inoculation of Test Portions
Replicates of each matrix varied by food or surface type and were inoculated with Lactobacillus fermentum ATCC 14931 or
Weissella viridescens ATCC 12706 at two target recovery levels on 3M Petrifilm AC Plates: a low level of 1–15 colony forming
units (CFU)/plate and a medium level of 15–75 CFU/plate. To prepare salad dressings and RTE salads for inoculation, the
suspensions of L. fermentum and W. viridescens were prepared in deMan, Rogasa, Sharpe broth (MRS) with 1.0% glucose. The
L. fermentum inoculum was incubated aerobically for 24 ± 2 hours at 35 ± 2°C and the W. viridescens inoculum was incubated
microaerophilically for 24 ± 2 hours at 35 ± 2°C. Twenty-five 1g sample portions were inoculated, mixed thoroughly, and held
for 48 ± 6 hours at 10°C to allow the organism to equilibrate within the matrix. For the analysis of RTE meats, L. fermentum
and W. viridescens inocula were prepared in MRS broth and incubated for 24 ± 2 hours at 35 ± 2°C. Twenty-five gram sample
portions were inoculated, mixed thoroughly, and held in a CO atmosphere to simulate packaging conditions for 48 ± 6 hours at
2
10°C. Following the equilibration step for all food matrices, 25 ± 1g test portions were diluted 1:10 with 0.1% peptone water.
If necessary, subsequent dilutions were made in 90mL of 0.1% peptone water to achieve the target recovery levels on PLAB.
Additionally, naturally contaminated brands of both RTE and raw meats were diluted in 0.1% peptone water until target recovery
levels were achieved.
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For the analysis of stainless steel environmental surfaces, sixteen 1" x 1" surface areas per organism were inoculated with a
dilution of a L. fermentum or W. viridescens broth culture. The L. fermentum inoculum was prepared using Brain Heart Infusion
Broth and incubated aerobically for 24 ± 2 hours at 35 ± 2°C. The W. viridescens inoculum was prepared in MRS and incubated
microaerophilically for 24 ± 2 hours at 35 ± 2°C. The inoculated surfaces were left to dry at ambient temperature for 18 ± 2
hours prior to sampling. For each organism, eight surface areas were sampled using environmental swabs pre-moistened with
Dey-Engley Neutralizing broth (D/E) and eight surface areas were sampled using environmental swabs pre-moistened with
Letheen Broth (LB). The surface areas were sampled in both horizontal and vertical motions. After sampling, the D/E swabs
were placed into a test tube containing 9mL of D/E and the LB swabs were placed into a test tube containing 9mL of LB.
Subsequent dilutions of these samples were prepared in 9mL test tubes of either D/E or LB to achieve the target recovery levels
on 3M™ Petrifilm™ Aerobic Count (AC) Plates.
Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M Petrifilm AC Plates Method
For the PLAB procedure, a 2.0mL aliquot of sample was transferred to a test tube containing 2.0mL of Lactic Bacteria Broth.
After vortexing the solution, a 1.0mL aliquot (0.5mL of sample and 0.5mL of Lactic Broth) was plated onto the 3M Petrifilm
AC Plate. PLAB plates were assayed in quadruplicate and incubated aerobically at 35 ± 2°C for food samples and 25 ± 2°C
for environmental samples for up to 72 ± 3 hours. Halos (acid zones) with and without associated colonies on PLAB plates
were enumerated after 24 ± 2 hours, 48 ± 3 hours, and 72 ± 3 hours of incubation. The presence of colonies with associated gas
bubbles were recorded after 72 ± 3 hours of incubation. For those RTE and raw meat samples that were naturally contaminated,
isolated colonies recovered from the PLAB plates were identified by transferring to tryptic soy agar (TSA) and incubated for 24
± 2 hours at 35 ± 2°C. Final organism identifications were achieved by the VITEK Anaerobe Identification (ANI) method.
Compendial and ISO Reference Methods
For the Compendial method procedure using All Purpose Tween agar plus 2% sucrose and bromocresol purple (APTS), 1.0mL
sample aliquots were pour plated in duplicate and incubated aerobically at 25 ± 2°C for up to 72 ± 3 hours. In addition to APTS
pour plates, salad dressing and stainless steel environmental samples were spread plated onto APTS using 0.25mL sample
aliquots. For the ISO reference method procedure using MRS plus bromocresol purple, 1.0mL sample aliquots were pour plated
in duplicate and incubated anaerobically at 35 ± 2°C. Two sets of MRS pour plates were prepared, one set incubated for 48 ± 3
hours and the other set incubated for 72 ± 3 hours. For each reference agar, colonies with and without halos were enumerated
after 48 ± 3 hours and 72 ± 3 hours. After the 72 hour enumeration, a representative colony from each sample plate was
transferred to MRS broth containing a Durham tube to detect gas production and was incubated at 35 ± 2°C for 5 to 7 days.
Results and Discussion
For the statistical analysis, a paired t-test for two sample means was calculated to analyze statistical differences between the PLAB
counts and reference agar counts at varying time points. The PLAB and reference agar time points used in the statistical analyses
are listed in Table B. The t-tests were calculated using counts across all brands and organisms for each inoculation level within the
matrices. Two procedures were used to obtain the values used in the statistical comparison. For the first method (Method 1), one
PLAB replicate from each brand and organism was compared to one reference agar replicate from each brand and organism. The
PLAB counts were converted to a count per milliliter by multiplying the 0.5mL value by a factor of two. The count per milliliter
values were converted to a Log values and compared to single reference agar counts converted to Log values in the t-test. For
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the second method (Method 2), all four PLAB replicates and both reference agar replicates were used in the t-test calculation. Two
3M Petrifilm AC Plate replicates were added together and the second two 3M Petrifilm AC Plate replicates were added together
to produce two count per milliliter values. The two counts calculated values were converted to Log values and averaged. The
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reference agar counts were converted to Log values and averaged. The averaged Log values from the PLAB method were
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compared to averaged Log values from each of the reference agars.
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Table B.
Method and Time Points Used in Statistical Analysis
48 Hour 3M™ Petrifilm™ AC Plate Counts vs. 48 Hour APTS Pour Plate Counts
48 Hour 3M™ Petrifilm™ AC Plate Counts vs. 72 Hour APTS Pour Plate Counts
48 Hour 3M™ Petrifilm™ AC Plate Counts vs. 72 Hour APTS Pour Plate Counts
72 Hour 3M™ Petrifilm™ AC Plate Counts vs. 72 Hour MRS Counts
72 Hour 3M™ Petrifilm™ AC Plate Counts vs. 72 Hour MRS Counts
Salad Dressings
For the 24 hour time point, colonies with associated halos were observed on the low level W. viridescens PLAB plates and
medium level W. viridescens PLAB plates had complete yellowing of the agar after 24 hours. The L. fermentum low and medium
level PLAB plates had no colonies or acid zones at 24 hours. Statistically comparable log values were observed comparing
48 hour 3M™ Petrifilm™ Aerobic Count (AC) Plate counts vs. 48 hour APTS pour plate counts at both levels using both
statistical procedures. A detailed list of t-test results for salad dressings is presented in Table 4 of Appendix 1.
RTE Salads
For the 24 hour time point, colonies with associated halos were observed on the low level W. viridescens PLAB plates. Medium
level L. fermentum and W. viridescens PLAB plates were completely yellow after 48 hours. At the 48 and 72 hour time point,
PLAB plates with colonies had associated halos and gas bubbles. Statistically comparable log values were observed comparing
72 hour 3M Petrifilm AC Plate counts vs. 72 hour MRS counts at the medium inoculation level using both statistical procedures.
Statistically comparable log values were observed comparing 72 hour 3M Petrifilm AC Plate counts vs. 72 hour APTS pour plate
counts at the medium inoculation level using t-test Method 2. A detailed list of t-test results for RTE salads is presented in Table
8 of Appendix 1.
RTE Meats
Colonies with associated halos were observed on PLAB plates from the naturally contaminated Private Selection Smokehouse
Hickory Ham. All low level PLAB plates had colonies with associated halos and gas production at the 48 hour time point. All
medium level PLAB plates were completely yellow at the 48 hour time point and had evidence of gas production. Statistically
comparable log values were calculated for the medium level 48 hour 3M Petrifilm AC Plate counts vs. 48 hour APTS pour plate
counts, 48 hour 3M Petrifilm AC Plate counts vs. 72 hour APTS pour plate counts, 72 hour 3M Petrifilm AC Plate counts vs.
72 hour APTS pour plate counts, and 72 hour 3M Petrifilm AC Plate counts vs. 72 hour MRS counts time points using both
t-test methods. L. acidophilus at an acceptable confidence level, L. jensenii at a very good confidence level, and L. cellobiosus
at an excellent confidence level were identified using the VITEK ANI method. A detailed list of t-test results for RTE meats is
presented in Table 11 of Appendix 1.
Raw Meats
At the 24 hour time point, PLAB plates were completely yellow with no associated colonies. At the 48 hour time point, PLAB
plates had colonies with associated gas production. APTS and MRS were overgrown with bacillus and an accurate enumeration
of lactic organisms could not be completed. L. acidophilus, L. jensenii, and L. casei were identified using the VITEK ANI
method at a low discrimination confidence level. A detailed list of PLAB counts for raw meats is presented in Table 12 of
Appendix 1. Due to overgrowth of the competing bacillus, no statistical comparisons were conducted for the raw meats.
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Stainless Steel Environmentals
Colonies with associated acid zones and gas production were observed on LAB plates at the 48 hour time period and complete
yellowing of the agars occurred at the 72 hour time point. Statistically comparable log values were calculated for the 48 hour
3M™ Petrifilm™ Aerobic Count (AC) Plate counts vs. 48 hour APTS pour plate counts comparison using both t-test methods. A
detailed list of t-test results for stainless steel environmental surface swabs is presented in Table 17 of Appendix 1.
Observations
The method comparison results obtained in the evaluation demonstrate the reliability of the PLAB procedure as an easy-to-use
method for the enumeration and detection of lactic acid organisms in a variety of foods and on stainless steel environmental
surfaces. The method requires a simple plating procedure and eliminates the lengthy gas production confirmation procedure
used in the reference methods. The PLAB method reduces the cost of sample preparation by eliminating the need for producing
and sterilizing reference method agars and eliminates costly materials needed to create and maintain an anaerobic environment
for MRS. The initial appearance of acid zones on PLAB is an accurate early indicator of the presence of lactic acid organisms
as acid zones were always observed surrounding typical red colonies. Colony associated gas production was observed on each
PLAB plate which is a distinct advantage over the reference procedures. The low inoculum level PLAB plates produced distinct
zones and colonies sooner than the medium level PLAB plates. It was often observed that the medium level did not have distinct
acid zones, instead the agar turned completely yellow and colonies were faint. When enumerating lactic organisms from matrices
with a high volume of non-target bacteria, the PLAB plates were easier to accurately enumerate compared to APTS. APTS often
had overgrowth of bacillus and other non- target bacteria, which made enumerating colonies with acid zones challenging. This
gives PLAB an advantage as only true lactic acid producing organisms and no bacillus was observed on 3M Petrifilm AC Plates.
3M and Petrifilm are trademarks of 3M.
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APPENDIX 1
Page 6
ur B 0 31 1 6 35 6 37 117 2 21 3 3 4 0 13 8 46 11 30 159 26 150 306 15 21 27 21 26 33 10
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0mL) 72 A 1 49 1 4 36 4 32 123 1 20 7 1 3 4 12 5 38 7 19 166 28 152 317 15 16 28 26 29 33 17
1.
MRS ( ur B 0 13 0 3 16 0 12 30 2 4 0 1 0 0 3 1 26 6 22 106 18 156 301 18 12 21 19 30 22 8
o
H
48 A 0 9 0 1 23 0 12 20 1 4 0 2 0 1 1 4 18 4 17 99 17 143 289 20 16 19 13 21 25 10
ur B 1 4 0 1 2 4 1 40 1 1 1 3 4 0 3 3 21 9 26 100 13 119 177 19 11 6 13 26 17 7
o
H
0mL) 72 A 3 8 3 0 3 1 2 50 0 0 1 1 4 0 1 1 20 6 18 110 11 116 216 10 13 8 11 25 20 9
1.
S (
APT our B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
H
8
4 A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
ur B 0 1 0 0 0 0 0 10 0 0 0 0 0 0 0 0 4 1 4 23 1 18 23 1 0 0 2 0 0 0
o
H
L) 2
5m 7 A 0 0 0 0 0 0 0 13 0 0 0 0 0 0 0 0 4 1 2 16 0 22 30 0 0 1 2 0 0 0
2
0.
S (
PT ur B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
A o
H
8
4 ate A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
c
mL) Repli D 0 0 0 0 5 1 6 50 0 0 1 1 0 0 0 0 11 2 8 58 4 39 96 1 2 1 9 4 4 3
5
0.
s (
Plate our* C 0 6 1 0 0 0 9 42 0 2 0 1 0 1 0 0 17 1 7 57 5 59 87 2 0 2 4 6 2 0
Count 72 H B 0 2 0 1 0 0 9 44 0 0 0 3 0 0 0 0 22 1 8 36 2 57 90 2 2 1 9 6 8 2
c
bi
ero A 0 2 0 0 2 2 15 45 0 0 1 0 1 1 0 0 13 1 6 54 4 63 93 2 0 1 4 3 6 3
A
™
m
etrifil D 0 0 0 0 3 0 3 20 0 0 0 0 0 0 0 0 4 0 7 17 0 11 80 0 0 1 3 0 0 0
P
™
h 3M our* C 0 0 0 0 0 0 3 24 0 0 0 0 0 0 0 0 1 0 2 21 1 20 83 0 0 0 1 2 1 0
m eria wit 48 H B 0 0 0 0 0 0 3 21 0 0 0 0 0 0 0 0 1 1 5 19 1 16 69 0 0 1 6 3 1 1
u ct
us ferment ctic Acid Ba DA 00 00 00 00 00 01 04 017 00 00 00 00 00 00 00 00 03 00 01 021 01 023 067 00 00 01 01 02 00 00
acill or La
ctob ure f our C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
a d H
— L Proce 24 B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
s c
ult obi
Res Aer A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
g
n
Salad DressiTable 1. Dressing Caesar 1 Caesar 2 LoFat Caesar 1 LoFat Caesar2 Ranch 1 Ranch 2 LoFat Ranch 1leveLoFat Ranch 2L woCatalina 1L Catalina 2 Blue Cheese 1 Blue Cheese 2 Blue Cheese 3 Zesty Italian 1 Zesty Italian 2 Caesar 1 Caesar 2 LoFat Caesar 1 LoFat Caesar 2 Ranch 1 Ranch 2leLoFat Ranch 1veL mLoFat Ranch 2uidCatalina 1eMCatalina 2 Blue Cheese 1 Blue Cheese 2 Blue Cheese 3 Zesty Italian 1 Zesty Italian 2 * Acid zones
Page 7
ur B 0 0 0 1 9 31 46 14 0 66 19 37 40 4 3 0 6 1 9 56 101 221 159 13 119 11 158 229 72 13
o
H
0mL) 72 A 0 0 0 1 10 29 50 11 0 78 20 36 29 5 1 0 6 2 10 48 88 206 177 12 128 6 139 216 59 19
1.
MRS ( ur B 0 2 1 2 10 17 43 16 2 73 24 16 20 8 4 0 6 6 7 49 93 188 186 9 126 8 180 221 66 18
o
H
48 A 0 1 2 1 11 20 39 18 0 70 26 20 27 7 3 0 9 10 12 43 81 200 189 11 121 8 166 200 48 15
ur B 0 3 2 7 17 23 51 49 2 86 24 31 44 4 3 0 11 10 21 73 126 226 270 8 513 NA 168 260 64 13
o
H
0mL) 72 A 0 6 2 3 18 19 44 55 0 89 29 28 41 4 4 0 7 9 20 69 115 231 254 8 570 NA 151 270 57 16
1.
APTS ( our B 0 2 2 6 14 23 50 47 1 86 22 28 40 4 3 0 9 10 17 73 120 219 266 8 513 NA 166 255 62 13
H
48 A 0 1 2 3 17 19 42 52 0 89 29 27 40 4 4 0 7 9 18 69 111 226 251 8 570 NA 146 266 57 16
ur B 0 0 0 0 3 7 5 17 0 21 4 5 2 0 0 0 0 0 0 17 27 89 93 0 126 4 38 48 21 0
o
H
L) 2
25m 7 A 0 0 0 0 1 3 8 12 0 23 1 4 2 0 0 0 0 0 0 13 31 64 89 0 117 9 38 42 17 0
0.
PTS ( ur B 0 0 0 0 3 5 5 17 0 19 2 5 2 0 0 0 0 0 0 0 23 84 93 0 121 4 38 47 20 0
A o
H
8
4 ate A 0 0 0 0 1 3 8 12 0 20 1 4 2 0 0 0 0 0 0 0 27 62 89 0 111 9 37 42 15 0
c
5mL) Repli D 0 0 0 0 2 11 20 29 0 40 1 8 8 2 0 0 0 0 0 3 58 123 169 3 210 18 71 98 25 3
0.
Plates ( our* C 0 0 0 0 1 9 16 35 0 45 2 11 14 0 0 0 0 0 0 6 67 136 187 3 213 24 72 84 28 0
Count 72 H B 0 0 0 0 0 1 12 25 1 45 8 12 5 3 0 0 0 0 0 5 65 117 154 4 203 32 66 73 17 2
c
bi
Aero A 0 0 1 0 1 5 21 36 0 28 6 11 9 2 0 0 0 0 0 3 65 123 135 3 171 34 45 60 25 3
™
m
etrifil D 0 0 0 0 2 10 20 29 0 40 1 8 8 2 0 0 0 0 0 1 53 120 167 3 210 18 69 95 25 0
P
h 3M™ our* C 0 0 0 0 1 8 16 35 0 45 2 11 14 0 0 0 0 0 0 5 66 134 187 3 213 21 71 82 27 0
eria wit 48 H B 0 0 0 0 0 1 12 25 1 45 8 10 5 3 0 0 0 0 0 2 63 113 154 4 200 29 65 72 15 0
ct
ens d Ba A 0 0 1 0 1 4 20 36 0 28 6 11 9 2 0 0 0 0 0 1 62 120 135 2 165 33 45 60 25 0
viridesc actic Aci D 0 0 0 0 0 7 10 8 0 8 0 6 4 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
— Weissella Procedure for L 24 Hour BC 00 00 00 00 00 06 86 98 10 48 20 43 44 20 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00
s c
ult obi
Res Aer A 0 0 0 0 0 3 8 7 0 4 3 5 4 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
g
n
Salad DressiTable 2. Dressing Caesar 1 Caesar 2 LoFat Caesar 1 LoFat Caesar2 Ranch 1 Ranch 2 LoFat Ranch 1leveLoFat Ranch 2L woCatalina 1L Catalina 2 Blue Cheese 1 Blue Cheese 2 Blue Cheese 3 Zesty Italian 1 Zesty Italian 2 Caesar 1 Caesar 2 LoFat Caesar 1 LoFat Caesar 2 Ranch 1 Ranch 2 leLoFat Ranch 1veL mLoFat Ranch 2uidCatalina 1eMCatalina 2 Blue Cheese 1 Blue Cheese 2 Blue Cheese 3 Zesty Italian 1 Zesty Italian 2 * Acid zones
Page 8
72 Hour MRS Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive P-Value 0.4190 0.0003 0.0290 0.0001 0.0003 0.3911 0.0001 0.0159 0.0000 0.0004
s
e
at
escens 72 Hour APTS Pour Plate Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive — Analysis of 4 Replic Ref Mean 0.4823 0.6699 0.6699 0.8265 0.8265 0.7657 1.4020 1.4020 1.4717 1.4717
d
Weissella viri 72 Hour APTS Spread Plate Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Method 2 Petrifilm Aerobic ™ unt Plate Mean 0.4128 0.4128 0.4752 0.4128 0.4752 0.8984 0.8984 1.1571 0.8984 1.1571
™ Co
M
™ 3
me
filat
Hour 3M Petri™ erobic Count Pl Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive P-Value 0.2887 0.0010 0.0148 0.0001 0.0002 0.3442 0.0002 0.0075 0.0000 0.0010
2 A
7
s
e
at
72 Hour MRS Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive 0.5 x 2 Replic Ref Mean 0.4759 0.6696 0.6696 0.8375 0.8375 0.7680 1.4198 1.4198 1.4655 1.4655
—
1
us fermentum 72 Hour ateAPTS Pour Plate Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Method 3M Petrifilm Aerobic ™™ Count Plate Mean 0.3673 0.3673 0.4355 0.3673 0.4355 0.9183 0.9183 1.1190 0.9183 1.1190
g Gas Production Results Lactobacill 72 Hour 3M Petrifilm 72 Hour ™™ Aerobic Count PlateAPTS Spread Pl PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive PositivePositive g T-Test Results Comparison m Aerobic Count Plate vs. 48 hour APTS m Aerobic Count Plate vs. 72 hour APTS m Aerobic Count Plate vs. 72 hour MRS m Aerobic Count Plate vs. 72 hour APTS m Aerobic Count Plate vs. 72 hour MRS m Aerobic Count Plate vs. 48 hour APTS m Aerobic Count Plate vs. 72 hour APTS m Aerobic Count Plate vs. 72 hour MRS m Aerobic Count Plate vs. 72 hour APTS m Aerobic Count Plate vs. 72 hour MRS
Salad Dressine 3. Salad Dressing Caesar 1 Caesar 2 LoFat Caesar 1 LoFat Caesar 2 Ranch 1 Ranch 2 LoFat Ranch 1 LoFat Ranch 2 Catalina 1 Catalina 2 Blue Cheese 1 Blue Cheese 2 Blue Cheese 3 Zesty Italian 1 Zesty Italian 2 Salad Dressine 4. 48 hour 3M Petrifil 48 hour 3M Petrifil 48 hour 3M Petrifil 72 hour 3M Petrifil 72 hour 3M Petrifil 48 hour 3M Petrifil 48 hour 3M Petrifil 48 hour 3M Petrifil 72 hour 3M Petrifil 72 hour 3M Petrifil
bl bl muluconI woL muluconI muideM
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Page 9
ur B 19 20 18 23 21 26 16 21 24 21 20 20 20 28 19 136 179 147 197 199 180 172 189 200 186 172 189 100 146 111
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2
mL) 7 A 16 14 21 28 24 19 20 27 22 21 18 26 21 23 16 141 168 141 200 187 166 188 176 206 201 146 161 109 129 119
0
1.
S (
MR ur B 19 15 19 23 16 12 20 27 28 24 27 22 22 26 20 140 199 166 226 201 201 191 188 218 158 161 192 108 166 106
o
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8
4 A 15 18 17 19 22 23 17 20 19 20 18 32 16 19 20 128 186 160 211 206 188 196 196 202 186 155 177 120 146 110
ur B 27 21 37 30 18 29 26 55 30 32 21 34 22 22 29 237 297 273 275 232 237 198 521 238 242 223 245 190 205 182
o
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2
mL) 7 A 20 20 20 39 24 19 24 63 31 27 19 30 28 19 22 217 303 288 297 269 266 223 539 242 226 201 208 221 206 209
0
1.
S (
APT ur B 21 19 32 29 17 25 25 46 27 31 19 32 19 21 28 226 291 259 267 263 229 219 497 232 201 191 178 210 195 201
o
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4 A 18 19 19 36 22 12 17 56 30 25 18 26 21 18 18 210 294 285 287 224 257 196 504 230 224 216 236 179 182 177
D 7 9 9 7 8 8 10 12 8 9 17 12 8 7 10 71 70 78 90 72 81 69 70 63 55 77 72 66 77 64
L)
0.5m ur* ate C 8 8 10 10 13 11 9 15 5 5 10 11 8 8 10 69 81 70 96 74 81 66 73 56 54 81 60 63 90 61
Plates ( 72 Ho Replic B 8 8 8 11 12 8 6 9 5 5 7 9 7 7 10 71 82 71 91 70 80 78 59 60 49 82 61 52 80 70
nt
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bic A 6 6 8 9 8 4 9 8 6 11 13 14 4 9 11 76 86 62 77 66 88 79 67 49 65 82 66 59 81 67
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film D 7 9 9 7 8 8 9 11 7 9 15 12 8 7 10 70 66 78 90 70 80 69 70 63 54 76 71 66 77 61
etri
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M™ * C 8 8 10 10 12 10 9 15 5 5 10 11 7 8 8 66 81 70 96 74 81 66 71 55 51 81 59 63 90 60
h 3 our
wit 8 H
eria 4 B 6 8 8 8 12 8 6 9 4 5 7 9 6 7 9 71 80 71 89 70 79 78 58 59 49 82 60 49 80 69
ct
a
B
cid A 6 5 8 9 8 4 9 8 5 11 13 14 4 9 11 76 86 62 74 66 88 79 66 49 64 79 66 59 81 66
A
c
m acti
ctobacillus fermentu Aerobic Procedure for L 24 Hour* BCD 471 624 443 572 484 263 448 7118 232 434 675 647 566 634 212 192628 403029 332631 362729 243130 282620 424027 393641 242019 172120 313026 222434 292630 371822 262223
a
L
—
s A 5 3 3 3 3 4 5 3 3 6 7 3 3 4 7 22 41 36 40 29 33 36 44 26 11 36 19 31 36 20
ult
s
RTE Salad Reable 5. Dressing Mustard Potato 1 Mustard Potato 2 Mustard Potato 3 American Potato 1 American Potato 2 American Potato 3 Macaroni 1leveMacaroni 2L woMacaroni 3L Bean 1 Bean 2 Bean 3 Coleslaw 1 Coleslaw 2 Coleslaw 3 Mustard Potato 1 Mustard Potato 2 Mustard Potato 3 American Potato 1 American Potato 2 American Potato 3 leMacaroni 1veL mMacaroni 2uidMacaroni 3eMBean 1 Bean 2 Bean 3 Coleslaw 1 Coleslaw 2 Coleslaw 3 Acid zones
T *
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