Detection of Injured Coliform Cells with the Sanita-kun Coliforms :

Transcription

1 Detection of Injured Coliform Cells with the Sanita-kun Coliforms : Comparison with VRB Agar Method. PTM license No Masashi Ushiyama, Mihoko Iwasaki, Hiroshi Morita Chisso Corporation, Yokohama Research Center

2 Sanita-kun Coliforms : Consists of a non-woven fabric to which a layer of microbial nutrients is deposited in a sheet. The nutrient sheet also contains X-gal, which is hydrolyzed by β-galactosidase from coliforms to form a visible blue dye.

3 forms :

4 Comparison with the VRB agar, FDA-BAM method, in natural contaminated foods Foods 50g Homogenize for 2 min 450mL PBS Dilution Sanitakun 1mL VRB agar:fda-bam 5 replicates VRB agar solution hr incubation Enumeration VRB: Red colonies Sanita-kun: Blue colonies Picking colonies Confirmation

5 Comparative Recovery of Naturally Contaminated Foods Coliform Counts (logcfu/g) Foods Sanita-kun VRB agar Raw Pork Raw Pork Raw Pork Raw Pork 3.76 a) 3.6 Raw Pork Raw Pork Frozen Pork 3.15 b) 2.45 Frozen Pork 3.00 b) 2.77 Frozen Pork Raw Chicken Raw Chicken Raw Chicken Smashed Sardine Smashed Sardine Smashed Sardine 3.11 a) 3.04 Sliced Perch Sliced Perch Sliced Perch Frozen Cod 3.04 b) 2.48 Frozen Cod 2.86 b) 2.46 Frozen Cod 2.38 b) 1.56 Sausages, Yakitori the Japanese brochette, Dried cod, Smoked salmon, Boiled soy bean, Frozen bean, Raisins, Ice cream, French roll, Coockie, Milk chocolate, Dog food, Dried Pet foods, Udon noodles. :<10cfu/g a)significantly higher than VRB Agar Method (p<0.05) b)significantly higher than VRB Agar Method (p<0.01) Sanita-kun (logcfu/g) Frozen Foods y = 0.69 x R 2 = 0.87 y = 1.04 x R 2 = 0.98 Frozen foods Non-frozen foods Non-frozen foods All foods VRB agar (logcfu/g)

6 Comparative Recovery of Naturally Contaminated Foods with Colony Confirmation Foods Coliform Count (log CFU/g) Sanita-kun Coliforms VRB Total Counts Confirmed Counts Raw Pork Raw Pork 5.13 a) 5.11 a) 5.05 Raw Ground Pork 3.61 b) Frozen Pork 3.15 b) 2.88 b) 2.20 Raw Chicken Raw Chicken Raw Chicken 4.79 b) Sliced Horse Mackerel Frozen Yellowtail 3.15 b) 3.04 b) 2.75 Frozen Yellowtail 2.57 b) Frozen Yellowtail 2.41 b) 2.32 b) 1.82 Frozen Bonito 2.41 b) 2.11 b) 1.08 Frozen & Sliced Suzuki 2.92 b) 2.76 b) 1.30 a) Significantly higher than VRB Agar Method (p<0.05) b) Significantly higher than VRB Agar Method (p<0.01)

7 Food Pork Chic ken Cod Comparative Recovery on 2 Week-Frozen Foods after Coliform Inoculation Inoculated E.coli IFO13500 C.diversus JCM1659 K.planticola IFO14939 K.pneumoniae IID5209 Target (cfu/g) Coliform (cfu/g) Sanita -kun VRB Agar Sanita -kun RSD FDA- BAM > > > > a) Significantly higher than VRB Agar Method (p<0.05) b) Significantly higher than VRB Agar Method (p<0.01) Fren ch Bea n Asp arag us Ice Crea m K.pneumoniae JCM166 2 E.aerogenes IFO K.oxytoca JCM166 5 E.cloacae IID977 > > > > <

8 Foods Repeatability or Precision 50g 50g 50g 50g 50g 26 items Homogenize 1mL Coliform Inoculation Sequential Preparation of Subsamples Dilution(1:10) 1mL 5 replicates VRB agar solution Incubate at 35 for 24hours Sanita-kun VRB Agar Sanita-kun VRB Agar Enumeration VRB: Red colonies Sanita-kun: Blue colonies

9 Categories Repeatability in Subsamples Foods Inoculation Target Level (CFU/g) Average Count (CFU/g) Sanita-kun RSD VRB Sanita-kun VRB Meat Raw Pork Klebsiella sp. No2 > Heat Sausage K.oxytoca JCM 1665 > Frozen Pork Klebsiella sp. No53 > Chicken Poultry Raw Klebsiella sp. No172 > Heat Yakitori K.pneumoniae IID 5209 > Frozen Chicken Enterobacter sp. No8 >

10 Repeatability in Subsamples Categories Frozen Dairies Foods Inoculation Heat Milk K.planticola IFO Low Moisture Bakery Dry Milk Choco -late Ice Cream Shredded cheese Bread Milk Chocolate Klebsiella sp. No52 K.pneumoniae IID 5209 E.aerogenes IFO Dry Coockie Klebsiella sp. No51 Klebsiella sp. No171 Target Level (CFU/mL) RSD Average Count (CFU/g) Sanita-kun VRB Sanita-kun VRB > > > > > >

11 Repeatability in Subsamples Yakitori, or Chicken Brochette 50g Inoculation 50g Inoculation 50g Inoculation 50g Inoculation 50g 1 ca.1000cfu 450mL PBS Stomacher Incubation 2 ca.1000cfu 450mL PBS Stomacher Incubation 3 ca.1000cfu 450mL PBS Stomacher Incubation 4 ca.1000cfu Preparation almost at the same time 450mL PBS Stomacher Subsample Preparation 1 by 1 in Sequence Colony enumeration (cfu/g) Incubation Sequence Sanita-kun VRB agar Inoculation 5 ca.1000cfu 450mL PBS Stomacher Incubation

12 Repeatability in Subsamples Averages of 26 Foods Relative colony count ( % ) % Sanita-kun, target: cfu) Sanita-kun, target:>100cfu VRB Agar, target:>100cfu VRB Aar. target:50-100cfu Sequence order

13 Effect of waiting time after sample dilution Coliforms in PBS Cfu/g Inoculation Min after Klebsiella pneumoniae IID5209 Enterobacter cloacae IFO13534 Escherichia coli IID5208 Sanita-kun VRB agar Sanita-kun VRB agar Sanita-kun VRB agar E.aerogenes IFO13534 inoculated in foods Cfu/g Foods Min after Tuna sashimi Sanita-kun VRB agar Shredded cheese Sanita-kun VRB agar Pork Sanita-kun VRB agar

14 Conclusions Sanita-kun Coliforms detected more colonies in frozen foods than VRB agar. More freeze-injured cells are recovered in the Sanita-kun sheet than in the VRB agar. Sanita-kun Coliforms is less influenced with a sample preparation process than VRB agar. Chisso Corporation sanita-kun@chisso.co.jp Web: