CERTIFICATION

AOAC Research Institute

Performance Tested MethodsSM
Certificate No.
092301
The AOAC Research Institute hereby certifies the method known as:

InnovateTM RapiScreen TM Dairy Kit

manufactured by
Hygiena LLC
941 Avenida Acaso,
Camarillo, CA
USA
This method has been evaluated in the AOAC Research Institute Performance Tested MethodsSM Program and found to perform as stated in the applicability of the
method. This certificate indicates an AOAC Research Institute Certification Mark License Agreement has been executed which authorizes the manufacturer to display the
AOAC Research Institute Performance Tested Methods SM certification mark on the above-mentioned method for the period below. Renewal may be granted by the
Expiration Date under the rules stated in the licensing agreement.

Issue Date September 12, 2023

Scott Coates, Senior Director Expiration Date December 31, 2023
Signature for AOAC Research Institute

2275 Research Blvd., Ste. 300, Rockville, Maryland, USA Telephone: +1-301-924-7077 Fax: +1-301-924-7089
Internet e-mail: aoacri@aoac.org * World Wide Web Site: http://www.aoac.org
AUTHORS SUBMITTING COMPANY
Benard Linke, Lucas Kemp, Romei Velasco, and Mathew Lovesmith Hygiena International LTD
8 Woodshots Meadow
Croxley Park
Watford, Hertfordshire, WD18 8YU

METHOD NAME CATALOG NUMBERS

InnovateTM RapiScreen TM Dairy Kit KIT4001 Ref. no. 82050 (Innovate RapiScreen Dairy 5000 Test Kit), Kit 4000 Ref.
no. 82010 (Innovate RapiScreen Dairy 1000 Test Kit), KIT4015 Ref. no. 1311250B
RapiScreen Dairy (25000) for Autosampler-Bar-Coded.

INDEPENDENT LABORATORY AOAC EXPERTS AND PEER REVIEWERS

Q Laboratories Thomas Hammack1, James Agin2, Elliot Ryser3
1930 Radcliff Drive 1 Food and Drug Administration, Center for Food Safety and Applied Nutrition,

Cincinnati, OH 45204 Maryland, USA

2
Independent Consultant, Ohio Department of Health (Retired), Ohio, USA
3 Michigan State University (Retired), Michigan, USA

APPLICABILITY OF METHOD REFERENCE METHODS

Target organism – Mesophilic microorganisms. American Public Health Association (APHA) Standard Methods for the
Examination of Dairy Products (SMEDP) Chapter 6: Microbial Count Methods,
Matrixes – ultra-high temperature (UHT) bovine milk, UHT plant-based 17th edition, 2004 (2)
drink (almond drink), half and half (10% fat), protein-based drink
(casein), extended shelf life (ESL) plant-based drink (oat drink). U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual
(BAM): Chapter 3, Aerobic Plate count, U.S. Food and Drug Administration.
Performance claims – The study data were unable to detect a statistical April 2001. (3)
difference in results between the Innovate System RapiScreen Dairy Kit
and the culture-based reference methods. The method can reliably U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual
detect microbial contamination in 7 days, as compared to a 15-day (BAM): Chapter 16, Clostridium perfringens, U.S. Food and Drug
reference method, and can detect contamination in some products in as Administration. April 2001. (4)
few as 3 days.
Compendium of Methods for the Examination of Food Products: Chapter 19:
Lactic Acid Bacteria (no date) (5)

ORIGINAL CERTIFICATION DATE CERTIFICATION RENEWAL RECORD

September 11, 2023 New Approval 2023.

METHOD MODIFICATION RECORD SUMMARY OF MODIFICATION

NONE NONE

Under this AOAC Performance Tested MethodsSM License Number, 092301 Under this AOAC Performance Tested MethodsSM License Number, 092301
this method is distributed by: this method is distributed as:
NONE NONE

PRINCIPLE OF THE METHOD (1)

The Innovate™ System is based on an automated benchtop luminometer capable of high throughput screening. The Innovate System works exclusively with the
RapiScreen™ family. RapiScreen utilizes adenosine triphosphate (ATP) bioluminescence, where the luciferase enzyme catalyses the consumption of microbial ATP
to produce light. ATP bioluminescence can detect viable microorganisms with high sensitivity, providing an objective result much faster, as compared to visible
microbial growth on agar plates, resulting in a faster detection time. RapiScreen includes a sample treatment step to reduce non-microbial sources of ATP prior to
performing the standard bioluminescence assay. A lysis step then releases microbial ATP for the bioluminescence reaction. The assay evaluated in this study is
RapiScreen Dairy.
Hygiena InnovateTM RapiScreen TM Dairy Kit, AOAC Research Institute Performance Tested MethodsSM certification number 092301

DISCUSSION OF THE VALIDATION STUDY (1)

The Innovate System luminometer using the RapiScreen Dairy Kit, is an easy and rapid way to detect the presence of microbial ATP in dairy and dairy alternative
products. It is intended to be used to provide quicker results than standard plating methods. The Innovate System RapiScreen Dairy can reliably detect low
concentrations of microbial ATP. The validation study confirmed that the Innovate System RapiScreen Dairy Kit can detect contaminated product packs in 7 days
or less, depending on the product, with results that are equivalent to the 15-day reference method requirement.
Time to detection is based upon both the matrix type used and the growth rate of the organisms. The microorganism used in this study panel were stressed prior
to inoculation, leading to the conclusion that healthy organisms could be detected quicker. In the inclusivity study, the Innovate System detected various types of
microorganisms, including Gram-positive and Gram-negative bacteria, yeasts and moulds. The RLU signal varied between organisms due to differences in
phenotype and growth rate. The method uses product-specific sterile RLU thresholds based on background ATP to determine when the organism has populated
the sample. The method was able to detect all 50 inclusivity microbes at concentrations less than 5 x 106 cfu/mL (2.7 x 105 per 50 µL test aliquot) per container.
Data obtained from matrix testing studies, which were closer to the designed use of the Innovate method than the inclusivity study, further supports this claim.
For all tested matrixes, the Innovate System RapiScreen Dairy method produced a 100% detection rate for the high positive spike level, low fractionally positive
results (5–15 positives) and negative results for the control containers. The Innovate System method was at least twice as fast, when 7-day incubation is
considered, when compared to the 15-day result of the reference methods.
In order to optimise the probability of contamination detection, the Innovate method must be validated for the specific product and incubation period for the
most likely microbial contaminants. The customer might also choose to validate an incubation period shorter than 7 days, as a shorter period might be sufficient
to detect high and/or fractional contaminants in certain matrixes. All positive results on the Innovate System method were significantly above the threshold
obtained from the matrixes’ RLU baselines. Based on this study, the Innovate System using RapiScreen Dairy Kit detected microbial contaminants in the examined
matrixes at least 10 days quicker than the reference method for the matrixes.
The Independent Matrix Study performed with the Innovate System RapiScreen Dairy Kit further substantiates the claim, having achieved successful detection of
microbial contamination in UHT bovine milk at and after 24 h of incubation. Using POD analysis, no statistically significant differences were observed between the
number of positive samples detected by the candidate method and the reference methods for all samples tested after 24 h of incubation. The RapiScreen Dairy Kit
combined with the Innovate System luminometer instrument and software provides a simple and rapid method for determining the commercial sterility of
manufactured products. The reagents are easy to prepare by adding a bottle of diluent to a bottle of lyophilized reagent. The software is intuitive and allows for
ease of use of the Innovate System instrument. Cleaning and maintaining the Innovate System instrument is convenient as the instrument has a self-cleaning wash
feature.
Further studies of robustness, instrument variation, product consistency and stability all achieved the required fractional levels of between 3 and 7 positives out of
10 and showed detection to be consistent across all conditions, independent of the examined organisms or variables introduced to the condition. Hence, the
Innovate System method is sensitive and robust, with the RapiScreen Dairy Kit reagents performing consistently across their shelf life. The Instrument Variation
study detected no variation in performance across three different luminometers that could affect the detection of a contaminant.

Table 2: RapiScreen Dairy Kit inclusivity results of 50 organisms. Readings were performed on the Innovate System and (+) and (-) results were given based on
broth/diluent control RLU thresholds. (1)
Candidate Method
No. Genus Species Source Origin Result cfu/sample aliquote
1 Alicyclobacillus acidoterrestris ATCCa 49025 Soil + 85,000
2 Aspergillus niger Wild Typeb Air isolate + 2,000
3 Bacillus cereus ATCC 11778 Unknown + 43,000
4 Bacillus coagulans NCTCc 3993 Soil + 30,000
5 Bacillus licheniformis Wild Type Plant-based drink + 268,000
6 Bacillus pumilus Wild Type Plant-based drink + 56,000
7 Bacillus spizizenii NCTC 10400 Unknown + 35,750
8 Bacillus subtilis ATCC 6051 Unknown + 119,000
9 Bacillus thuringiensis Wild Type Plant-based drink + 69,000
10 Byssochlamys fulva ATCC 10099 Bottled fruit + 22,500
11 Candida albicans ATCC 10231 Bronchomycosis + 52,250
12 Candida orthopsilosis NCPFd 8798 Human + 69,250
13 Cellulosimicrobium cellulans NCTC 13518 Human + 26,800
14 Citrobacter freundii NCTC 9750 Unknown + 13,600
15 Clostridium perfringens NCTC 8237 Water + 223,000
16 Clostridium sporogenes ATCC 7955 Unknown + 104,000
17 Corynebacterium renale ATCC 10848 Human + 35,000
18 Cronobacter sakazakii ATCC 29544 Human + 184,800
19 Dekkera bruxellensis ATCC 36234 Belgian stout + 40,000
20 Enterobacter aerogenes ATCC 13048 Sputum + 44,500
21 Enterobacter cloacae NCTC 10005 Spinal fluid + 58,800
22 Enterococcus faecalis ATCC 19433 Piglet feces + 35,700
23 Escherichia coli ATCC 8739 Feces + 18,800
24 Geobacillus stearothermophilus ATCC 7953 Unknown + 2,500
25 Kluyveromyces lactis ATCC 20185 Cheese + 23,500
26 Kluyveromyces marxianus NCTC 3106 Creamery + 35,750
27 Lactobacillus fermentum ATCC 9338 Milk + 13,900
28 Lactobacillus fructivorans ATCC 8288 Unknown + 243,000
29 Lactobacillus lactis ATCC 19435 Cheese + 7,500
30 Lactobacillus paracasei Wild Type Ketchup + 9,8500
31 Lactococcus lactis ATCC 11454 Milk + 9,700
32 Leuconostoc mesenteroides ATCC 8293 Olives + 7,900
33 Listeria innocua ATCC 33090 Cow brain + 14,300
34 Listeria monocytogenes ATCC 7644 Human + 17,400
Hygiena InnovateTM RapiScreen TM Dairy Kit, AOAC Research Institute Performance Tested MethodsSM certification number 092301

35 Micrococcus luteus ATCC 4698 Human + 53,500

36 Penicillium chrysogenum ATCC 10106 Cheese + 8,000
37 Pseudomonas aeruginosa ATCC 9027 Ear infection + 17,500
38 Pseudomonas fluorescens ATCC 13525 Water + 16,200
39 Pseudomonas putida ATCC 49128 Clinical isolate + 23,000
40 Saccharomyces cerevisiae ATCC 9763 Distillery + 16,500
41 Saccharomyces kudriavzevii ATCC 2601 Unknown + 6,950
42 Salmonella Enteritidis ATCC 13076 Unknown + 26,200
43 Salmonella Newport NCTC 14032 Unknown + 22,500
44 Salmonella Typhimurium ATCC 14028 Chicken liver + 21,000
45 Staphylococcus aureus ATCC 6538 Human lesion + 12,700
46 Talaromyces pinophilus ATCC 36839 PVC + 800
47 Torulaspora delbrukeii ATCC 10662 Unknown + 26,500
48 Yarrowia lipolytica ATCC 9773 Butter + 8,000
49 Zygosaccharomyces parabailii ATCC 56075 Unknown + 52,500
50 Zygosaccharomyces rouxii ATCC 2623 Grape must + 13,925
a
American Type Culture Collection, Manassas, VA.
bWild type strains isolated at Hygiena LLC Research Laboratory, Camarillo, CA.
cNational Collection of Type Cultures, Porton Down, Salisbury, UK.
dNational Collection of Pathogenic Fungi, Porton Down, Salisbury, UK.
eSample aliquot is 50 µL.

Table 4: RapiScreen Dairy Kit results of the Spiked Matrices and Respective Strains Comparing the Candidate Method to the Reference Method. (The
Innovate System read timepoint shown is described in column 4). (1)
Spiked Candidate method Reference method
cfu per
Matrix Strain packa Day Nb xC PODC d 95% CI x PODR e 95% CI dPODC f 95% CI g
11 5 5 1.00 (0.57, 1) 5 1.00 (0.57, 1) 0 (-0.47, 0.47)
Pantoea
UHT bovine milk agglomerans 1.2 2 20 12 0.60 (0.39, 0.78) 13 0.65 (0.43, 0.82) -0.05 (-0.21, 0.11)
ATCCh 27155
0 5 0 0.00 (0, 0.43) 0 0.00 (0, 0.43) 0 (-0.47, 0.47)

588 5 5 1.00 (0.57, 1) 5 1.00 (0.57, 1) 0 (-0.47, 0.47)

Bacillus
ESL plant-based
coagulans 1 5 20 8 0.40 (0.22, 0.61) 10 0.50 (0.3, 0.7) -0.1 (-0.28, 0.08)
drink
ATCC 7050
0 5 0 0.00 (0, 0.43) 0 0.00 (0, 0.43) 0 (-0.47, 0.47)

6300 5 5 1.00 (0.57, 1) 5 1.00 (0.57, 1) 0 (-0.47, 0.47)

Clostridium
Half and half sporogenes 7 5 20 7 0.35 (0.18, 0.57) 7 0.35 (0.18, 0.57) 0 (-0.13, 0.13)
ATCC 7955
0 5 0 0.00 (0, 0.43) 0 0.00 (0, 0.43) 0 (-0.47, 0.47)

19000 5 5 1.00 (0.57, 1) 5 1.00 (0.57, 1) 0 (-0.47, 0.47)

Lactobacillus
Protein-based
fermentum 1 7 20 7 0.35 (0.18, 0.57) 7 0.35 (0.18, 0.57) 0 (-0.13, 0.13)
drink
ATCC 9338
0 5 0 0.00 (0, 0.43) 0 0.00 (0, 0.43) 0 (-0.47, 0.47)

9.6 5 5 1.00 (0.57, 1) 5 1.00 (0.57, 1) 0 (-0.47, 0.47)

Bacillus
UHT plant-based
subtilis 0.6 5 20 8 0.40 (0.22, 0.61) 8 0.40 (0.22, 0.61) 0 (-0.13, 0.13)
drink
ATCC 6051
0 5 0 0.00 (0, 0.43) 0 0.00 (0, 0.43) 0 (-0.47, 0.47)

2-10 5 5 1.00 (0.57, 1) 5 1.00 (0.57, 1) 0.00 (-0.47, 0.47)

Pantoea
UHT bovine milk i
agglomerans 0.2-2 2 20 8 0.40 (0.22, 0.61) 8 0.35 (0.22, 0.61) 0.00 (-0.13, 0.13)
CCUG 58262
0 5 0 0.00 (0, 0.43) 0 0.20 (0, 0.43) 0.00 (-0.47, 0.47)
a cfu = colony forming units applied to each package.
b
N = number of test portions.
c X = number of positive test portions.
d POD = Candidate Method presumptive results confirmed positive divided by the total number of trials.
C
e POD = Reference Method positive results divided by the total number of trials.
R
f dPOD = Difference between the Candidate Method results and Reference Method results POD values.
C
g 95% CI = if the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.
h ATCC = American Type Culture Collection, Manassas, VA.
i Matrix tested in the independent laboratory, Q-Laboratories, Cincinnati, OH.
Hygiena InnovateTM RapiScreen TM Dairy Kit, AOAC Research Institute Performance Tested MethodsSM certification number 092301

REFERENCES CITED
1. Linke, B., Kemp, L., Velasco, R., and Lovesmith V., Validation of the Innovate™ RapiScreen™ Dairy Kit for Detection of Mesophilic Microorganisms in Ultra-
High Temperature (UHT) Bovine Milke, UHT Plant-Based Drink, Half and Half, Protein-Based Drink, and Extended Shelf-life Plant-Based Drink, AOAC
Performance Tested MethodsSM certification number 092301. Approved September 2023.
2. American Public Health Association (APHA) Standard Methods for the Examination of Dairy Products (SMEDP) Chapter 6: Microbial Count Methods, 17th
edition, 2004
3. U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM): Chapter 3, Aerobic Plate count, U.S. Food and Drug Administration. April
2001. Available at: https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count
4. U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM): Chapter 16, Clostridium perfringens, U.S. Food and Drug Administration.
April 2001. Available at: https://www.fda.gov/food/laboratory-methods-food/bam-chapter-16-clostridium-perfringens
5. Compendium of Methods for the Examination of Food Products: Chapter 19: Lactic Acid Bacteria (no date)