Performance Evaluation of Ready-to-Use Culture Media, Easy Plate AC for Enumeration ofAerobic Plate Count in a Broad Range of Foods, Environmental Samples and Pet Foods
Shinichiro Sugiura1, Suzanne J. Jordan2, Kentaro Takenaka3, Mai Shimizu3 and Takeo Suzuki3(1)Kikkoman Biochemifa Company, (2)Campden BRI, (3)Kikkoman Corporation

2. Summary

1. Introduction

E-AC was shown to be comparative to the ISO reference method 48331 :2013 for a broad range of foods, environmental samples and pet foods.

・For the enumeration of an aerobic count, plate
count agar (PCA) is traditionally used according
to ISO 4833-1 (2013)

E-AC is found to be

1. Reliable method to enumerate aerobic plate count✓Comparative to the ISO 4833-1:2013 method
2. Applicable to a broad range of foods, environmental
samples and pet foods✓ With reasonable repeatability and accuracy
3. Provide results in 48h, a saving of 24h compared withtraditional PCA
4. Compatible with foods containing Bacillus spp. that are
liable to spreading

・A type of Ready-to-use (RTU) media, Easy
Plate AC (E-AC) (Kikkoman BiochemifaCompany) can be alternatively used to provide
many benefits including reduced time toresult, simplicity of use and is plastic-saving
compared to PCA.

3. Methods & Results
The Method validation study was done according to ISO 161402:2016, using ISO 4833-1:2013 as the reference method. Analysis
with E-AC was performed following manufacturer’s instructions. The
incubation condition used for the study are displayed in Table 1.

Table 1. Incubation conditions used for the study

MediaTemperature

TimePCA
E-AC

30℃
30℃

72h
48h

3.2. Accuracy profile study

・A total of 210 artificially contaminated samples across 7 categories with
2 items per category were tested in this section. Each item used
was contaminated at 3 levels, with 5 replicates analyzed per level.
→ All 7 categories passed the 0.5log acceptability limits or therecalculated limits.
Additional work to determine the impact of E-AC on the growth of Bacillus
spp. was also performed

3.3. Evaluating the spread of colonies

3.3.1. Analysis with foods containing Bacillus spp.

3.1. Relative trueness study

・A total of 105 samples across 7 categories was tested. Eachcategory contained 3 types and 15 items per category. Details ofcategories and types used are shown in Table 2.
→ No significant difference between E-AC and the ISO method
was noted as revealed in the scatter plot displayed in Figure 1below.

・Six foods were plated to E-AC and PCA→ Colonies spread over the surface of PCA (Almost unable to countcolonies)→ The spread was prevented on E-AC.

Figure 2. Evaluating the spread of colonies
FlourFrozen spinach
Garam masala

Table 2. Categories and types
tested in the relative trueness study

Raw milk and dairy products

Milk and dairyPasteurized milk and milk based products(raw and heat processed)

Dry milk products

Fishery(raw, ready-to-eat, ready-to-reheatand ready-to-cook)

Produce and fruits(fresh and processed)

Multi-component foods ormeal components

Raw fish
RTE/RTC/RTRH fish and seafoods
Crustaceans
Cut ready-to-eat vegetables/leafy greensand sprouts
Fresh fruit/Cut RTE fruit and vegetableproducts
Heat treated fruit and vegetables
Composite foods with substantial raw
ingredients
RTRH/RTE foods (chilled, frozen)
Mayonnaise based deli-salads
Raw poultry and meat cuts

Meat and poultry

Raw processed meat

(raw and ready to cook)

RTC processed poultry
Dry Food

Pet food and animal feed

Wet food (raw and canned)
Animal feeds (poultry and fish)
Surfaces (wipes, swabs)

Environmental samples

Process water(food or feed production)

Dusts

Figure 1. Correlation between E-AC and the ISO method
10Noodle

Rice ballSeaweed with perilla

3.3.2. Evaluating the spread of Bacillus spp. pure cultures・Six spreading colonies of 3.3.1 were identified with 16S rRNA
sequencing.
→ All of them were identified as Bacillus spp.
・Isolates were plated to PCA, E-AC and another type of RTU media (RTU1)
after pure culture.
→ The spread was prevented on E-AC for all species while not on PCA
and RTU1 for some species.

Table 3. Evaluating the spread of Bacillus. spp.
with pure culture
Organism

Top hits

OriginBacillus sp.

B. cabrialesii,B. inaquosorum

Garammasala

B. velezensis

B. Velezensis

Flour

B. cereusgroup

B. pseudomycoides

FrozenspinachBacillus sp.

SeaweedB. amyloliquefaciens,withB. velezensis
perilla

PCA×E-AC✓RTU1×

9log10 CFU/g E-AC8765432Bacillus sp.

B. subtilis,
B. stercoris

B. altitudinis

B. aerius,
B. altitudinis,
B. aerophilus,
B. stratosphericus

100123456log10 CFU/g PCA

78910×

✓××

✓××

✓×

Rice ball×✓✓

NoodleSpreadmodestly✓✓“✓” indicates “easy to count” while “×” indicates “difficult or unable to count”