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Yeast strains specified for use in this independent validation study were obtained from Microbiologics,
1
Inc. One technician prepared samples and a second technician performed the assays on “blind” coded
2
samples.
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All samples were fully randomized and labeled with a code so that the analyst performing the assays
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was unaware of the level or type of bacterial culture present.
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Test Organisms
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Trichosporon mucoides
(ATCC ® 201382 ™ ) and
Kluyveromyces lactis
(ATCC 8563) were grown
7
individually in YM broth. Serial dilutions were prepared in tubes of sterile distilled water diluent to
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achieve an inoculation level of 10-100, 100-1000, 1000-10000 CFU/g in each of the foods.
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Product Inoculation
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Frozen beef patties were inoculated in bulk with
T. mucoides
(Table 30)
to have a target of 10-100, 100-
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1000.1000-10000 CFU/g of the culture in the product, thoroughly mixed, and stored at -20°C for 2
13
weeks. The yogurt was inoculated
K. lactis
(Table 29) for a target of 10000-CFU/g of culture, mixed and
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diluted with uninoculated yogurt for 2 additional target levels of culture of 10-100 CFU/g and 100-1000
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CFU/g.
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17
Aerobic Plate Count
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One 25g sample of each lot of uninoculated frozen ground beef patties and yogurt was analyzed
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for aerobic plate count (AOAC 990.12).
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21
Microbial Analysis
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A 1:10 dilution of each 25g sample was prepared with 225ml of 0.1% peptone. The samples were
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homogenized for 2 min and the sample diluted with 0.1% peptone for 5 additional dilutions. One ml of
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each dilution was plated onto duplicate plates of RYM. The plates were then be incubated at 25 ± 1°C
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for 60h. The plates were read after 48 and 60h of incubation. In addition, 100 µl of each dilution was
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plated onto triplicate plates of DRBC with 1ml of sample plated across 3 plates for the 1/10 dilution and
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incubated at 25 ± 1°C for 5-7 days.
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For each matrix, five samples were analyzed per replicate, 5 at 10-100 CFU/g, 5 at 100-1000 CFU/g,
29
and 5 at 1000-10000 CFU/g, and 5 un-inoculated control samples.
30
31
The yogurt and frozen beef patties had <1 Log CFU/g of yeast and mold in the uninoculated product
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AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-16D/ PTM Report
ERP Use Only - December 2014