NFI-PTM 37-2014

Department of Livestock Development

Proficiency Testing Report Proficiency Testing Program for DLD Designated Laboratories

Round 2/2014

Proficiency Testing Provider

Division of Proficiency Testing Services, National Food Institute, Thailand

Microbiological Analysis

NFI-PTM 37-2014: Yeast and Mold Count (cfu/g) in Corn Powder

May 2014

NFI-PTM 37-2014

หนา ข

CONTENTS SUMMARY A 1. INTRODUCTION 1.1 Proficiency Testing 1 1.2 Yeast and Mold 1 2. TEST MATERIALS 2.1 Sample Preparation 2 2.2 Homogeneity 2 2.3 Stability 3 2.4 Sample Distribution 3 3. REPORTING 4

4. STATISTICAL EVALUATION OF RESULTS 4.1 Calculation of the Assigned value, X and Standard uncertainty of

the assigned value, uX

4

4.2 Calculation of standard deviation for proficiency assessment,σ̂ 5

4.3 Calculation of robust Z-Score 5 4.4 Z-Score assessment criteria 5 5. REFERENCES 5

6. RESULTS Table 1 Result summary of participants’ Z-Score for yeast and mold count (cfu/g)

in corn powder 6

Table 2 Statistics Summary 9 Figure 1 Z-Score values for yeast and mold count (cfu/g) in corn powder 10

7. TECHNICAL COMMENTS 11

8. CONTACT DETAILS 11 9. APPENDIX

Table 3 Homogeneity and stability data 12 Table 4 Technical information in relation to the methods 13 COMMITTEE 14

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Page A

SUMMARY

This summarizes the microbiological proficiency testing program of NFI-PTM 37-2014: Yeast and Mold Count (cfu/g) in Corn Powder, under the project “Proficiency Testing Program for DLD Designated Laboratories, Round 2/2014”.

There were 43 laboratories participating in this program. The test materials were quality controlled to ensure that consistent levels were achieved throughout the batch by conducting homogeneity test prior to dispatch. Stability testing was conducted at the end of shelf life which was the deadline for all laboratories to run the test – to ensure the integrity of the test materials.

Forty three laboratories submitted their results which were assessed statistically in the form of a robust Z-Score for performance evaluation for each laboratory. The overall competency evaluation was that there were 42 laboratories (97.7%) had satisfactory performance with ⏐Z⏐≤2, and 1 laboratory (2.3%) had questionable performance with 2 < ⏐Z⏐< 3.

This proficiency testing program accomplished with smooth operation as expected, and every participating laboratory benefits and acquires valuable information for advancement whatever the performance result is.

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1. INTRODUCTION

1.1 Proficiency Testing

Proficiency testing (PT) is a valuable tool for quality assurance of testing and measurement. It is a requirement of accreditation to ISO/IEC 17025 that laboratories take part in proficiency testing schemes. PT involves the use of interlaboratory comparisons for determination of laboratory performance with the purpose of producing laboratory results of greater reliability. Whatever the performance result is, every participating laboratory benefits and acquires valuable information for further advancement in all degree of their current performance.

For the improvement and satisfaction to the requirement of ISO/IEC 17025 of laboratories, hence the Division of Proficiency Testing Services, Department of Laboratory Services, National Food Institute (NFI) provides PT program to follow the international standard ISO/IEC 17043:2010, Conformity Assessment – General Requirements for Proficiency Testing. This program was named “Microbiological Analysis NFI-PTM 37-2014: Yeast and Mold Count (cfu/g) in Corn Powder” with the focus on Yeast and Mold since it is cause various degrees of deterioration and decomposition of foods.

To ensure performance confidentiality of all participants, a series of Laboratory Number is used in place of participant’s name. A unique Laboratory Number is assigned to each laboratory.

NFI offers Internet access for proficiency testing at ‘http://pt.nfi.or.th’ for fast, convenient and easy access for participants to register, submit form / data / test result / request, view other information, and communicate with the provider. All participants are encouraged to utilize all on-line features and more for complete and clear action for their own advantage.

1.2 Yeast and Mold

The large and diverse group of microscopic foodborne yeasts and molds includes several hundred species. The ability of these organisms to attack many foods is due in large part to their relatively versatile environmental requirements. Although the majority of yeasts and molds are obligate aerobes (require free oxygen for growth), their acid/alkaline requirement for growth is quite broad, ranging from pH 2 to above pH 9. Their temperature range (10-35°C) is also broad, with a few species capable of growth below or above this range. Moisture requirements of foodborne molds are

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relatively low; most species can grow at a water activity (aw) of 0.85 or less, although yeasts generally require a higher water activity.

Both yeasts and molds cause various degrees of deterioration and decomposition of foods. They can invade and grow on virtually any type of food at any time; they invade crops such as grains, nuts, beans, and fruits in fields before harvesting and during storage. They also grow on processed foods and food mixtures. Their detectability in or on foods depends on food type, organisms involved, and degree of invasion; the contaminated food may be slightly blemished, severely blemished, or completely decomposed, with the actual growth manifested by rot spots of various sizes and colors, unsightly scabs, slime, white cottony mycelium, or highly colored sporulating mold.

Several foodborne molds, and possibly yeasts, may also be hazardous to human or animal health because of their ability to produce toxic metabolites known as mycotoxins. Most mycotoxins are stable compounds that are not destroyed during food processing or home cooking. Even though the generating organisms may not survive food preparation, the preformed toxin may still be present.

2. TEST MATERIALS

2.1 Sample Preparation

Test materials were produced in bulk by blending dried kernel corn, powdering, and mixing it well. Tight-sealing-cap plastic tubes containing 10 grams of the corn powder were prepared, and all the tubes with numbers labeled were stored at 4-10°C for homogeneity test and other steps.

2.2 Homogeneity

To insure that consistent levels of organisms were achieved throughout the batch of test materials, homogeneity test was conducted on 2 May 2014. The random number generator from the website ‘random.org’ was used to randomize the order of the samples. Ten out of all the tubes were randomly picked for homogeneity test by carrying out yeast and mold count (cfu/g) in duplicate prior to dispatch.

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2.3 Stability

To ensure the constancy of the test materials not being diminished over time and the transport effect, stability testing was conducted at the end of shelf life. Therefore five sets of the test materials at NFI were packaged using the same types of materials (boxes, ice packs, etc.) and conditions (temperature, length of time, etc.) as those delivered; leaving the selected-randomly sample in the box for 24 hours, and then keeping at 4-10 °C until the final test date. On 23 May 2014, the test materials were examined in duplicate for enumeration of yeast and mold count (cfu/g).

The work on homogeneity and stability tests was conducted by the supporting laboratory – Division of Microbiological Laboratory, National Food Institute, (ISO/IEC 17025: DMSC Acc. No. 1005/42). The homogeneity and stability data were shown in Table 3 in Appendix.

2.4 Sample Distribution

Upon completion of homogeneity test, foam boxes of test materials with ice packs, and documents were dispatched on 14 May 2014 to each participant by an express logistics company with 24-hour guaranteed delivery. Documents enclosed in the box were as follows:

1) Letter of Introduction 2) Test Instruction Sheet 3) Receipt Form 4) Results Form 5) Technical Form 6) On-line Instruction Sheet Note: - Filling on-line preferred for 3), 4) and 5). - Using test methods (materials, procedures, conditions, etc.) consistent with each

laboratory routine practices. - One tube of test material delivered for each participant.

Participants were required to report and submit their enumeration of the test material in cfu/g by the closing date – “30 May 2014”.

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3. REPORTING

Having finished the calculation of the test result, each participating laboratory then submitted its test result by the closing date for analysis, and for the summary report to be made. With all these data, together with those received through PT provider’s quality control, statistical analysis for homogeneity test, stability test, and others were carried out according to the standard ISO 13528:2005.

The summary results are given in Table 1. Table 2 summarizes the statistics calculated. Technical data of methods used by each laboratory in relation to accreditation, reference, and others are summarized in Table 4 in Appendix.

Interim report and final report could be uploaded to the website pt.nfi.or.th within two weeks and one month respectively after the closing date, while hard copies of interim and final report could be sent to participants with no Internet access by registered mail.

4. STATISTICAL EVALUATION OF RESULTS

Statistical use for this proficiency testing program is robust Z-Score that is based on ISO 13528:2005.

4.1 Calculation of the Assigned value, X and Standard uncertainty of the assigned value, uX

4.1.1 Assigned value, X (assigned value for proficiency assessment) was set as the consensus of the results submitted by participants. The procedure used to derive this consensus involved some or all of the following:

4.1.1.1 Log transformation of participant results from cfu/g to log10cfu/g. 4.1.1.2 Removal of data that was not considered valid. 4.1.1.3 Derivation of the robust mean (x*) of the final data set using a robust

statistical procedure that calculate by Algorithm A. 4.1.1.4 Assessment of the standard uncertainty of the assigned value, uX .

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p

sux

*

25.1 ×=

where s* is robust standard deviation calculated by Algorithm A and p is number of result.

4.2 Calculation of standard deviation for proficiency assessment, σ̂

Standard deviation for proficiency assessment used for this proficiency testing program is robust standard deviation (s*) that calculate by Algorithm A.

4.3 Calculation of robust Z-Score

Where x is participant’s reported result,

X is assigned value and σ̂ is standard deviation for proficiency assessment.

4.4 Z- Score assessment criteria ⏐Z⏐ ≤ 2 is Satisfactory

2 <⏐Z⏐ < 3 is Questionable ⏐Z⏐ ≥ 3 is Unsatisfactory

5. REFERENCES

ISO/IEC 17043:2010. Conformity assessment – general requirements for proficiency testing.

ISO 13528:2005. Statistical methods for use in proficiency testing by interlaboratory comparisons.

T. Valerie, M. E. Stack, P. B. Mislivec, H. A. Koch and R. Bandler. 1998. Chapter 18 – Yeasts, Molds and Mycotoxins, in the Bacteriological Analytical Manual – Online (April 2001), Food and Drug Administration.

http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm071435.htm (June 2014).

Z-Score = ( log10 x – log10 X )

σ̂

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6. RESULTS

Table 1 Result summary of participants’ Z-Score for yeast and mold count (cfu/g) in corn powder

Laboratory Number

Result cfu/g

Result Log 10 cfu/g

Z-Score

1 190000 5.279 1.8

2 6.5E+04 4.813 -0.7

3 7.0E+04 4.845 -0.5

4 120,000 5.079 0.7

5 75000 4.875 -0.4

6 110000 5.041 0.5

7 2.27E+05 5.356 2.2

8 6.4E+04 4.806 -0.7

9 160,000 5.204 1.4

10 77000 4.886 -0.3

11 74000 4.869 -0.4

12 170000 5.230 1.5

13 2.0E+05 5.301 1.9

14 63000 4.799 -0.8

15 90000 4.954 0.1

16 130000 5.114 0.9

17 49000 4.690 -1.3

18 62000 4.792 -0.8

Note: Z-Score values outside the satisfactory range are shown in bold.

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Table 1 Result summary of participants’ Z-Score for yeast and mold count (cfu/g) in corn powder (continued)

Laboratory Number

Result cfu/g

Result Log 10 cfu/g

Z-Score

19 130000 5.114 0.9

20 36000 4.556 -2.0

21 130000 5.114 0.9

22 72000 4.857 -0.4

23 86000 4.934 0.0

24 82000 4.914 -0.1

25 69000 4.839 -0.5

26 59000 4.771 -0.9

27 7.4E+04 4.869 -0.4

28 6.0E+04 4.778 -0.9

29 5.0E+04 4.699 -1.3

30 100000 5.000 0.3

31 94000 4.973 0.2

32 40000 4.602 -1.8

33 140000 5.146 1.1

34 1.1E+05 5.041 0.5

35 1.2E+05 5.079 0.7

36 9.5E+04 4.978 0.2

Note: Z-Score values outside the satisfactory range are shown in bold.

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Table 1 Result summary of participants’ Z-Score for yeast and mold count (cfu/g) in corn powder (continued)

Laboratory Number

Result cfu/g

Result Log 10 cfu/g

Z-Score

37 8.2E+04 4.914 -0.1

38 100000 5.000 0.3

39 57000 4.756 -1.0

40 63,000 4.799 -0.8

41 5.6x104 4.748 -1.0

42 1.8x105 5.255 1.6

43 1.3x105 5.114 0.9

Note: Z-Score values outside the satisfactory range are shown in bold.

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Table 2 Statistics summary

Assigned Value Standard Deviation

for Proficiency Assessment

Number of result

Robust mean, x* (log10 cfu/g)

Robust standard deviation, s*

Standard uncertainty, uX

Derived from

σ̂

43 4.942 0.190 0.036 s* 0.190

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7. TECHNICAL COMMENTS

It is very important to find errors and correct them. For this proficiency testing program there are two main viewpoints to be considered by those in need of improvement: 7.1 Calculation of the test result and the data transfer The laboratory must carefully calculate the test result, e.g. multiplication of the dilution and checking of the data transfer before reporting.

7.2 Analytical technique The laboratories must be careful to all details in the testing process, such as mixing,

incubation temperature, and incubation time. NFI is pleased to provide further detail, and welcome any discussion regarding the proficiency testing.

8. CONTACT DETAILS

Upon receiving the final report, if there is any question regarding the proficiency testing assessment incurs, written notification should be submitted to the project coordinator by one of the following channels:

Mail: Project coordinator Division of Proficiency Testing Services, Department of Laboratory Services, National Food Institute, 2008 Soi Arun Ammarin 36, Arun Ammarin Rd., Bangyeekhan, Bangplad, Bangkok 10700 THAILAND.

Fax number: 0 2886 8088 ext. 5555 E-mail: pt@nfi.or.th Website: Contact-Us Form at http//:pt.nfi.or.th

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9. APPENDIX

Table 3 Homogeneity and stability data

Yeast and Mold (log10 cfu/g)*

Homogeneity (x) Stability (y) Sample No.

Replicate 1 Replicate 2 Sample No.

Replicate 1 Replicate 2

1 4.924 5.041 1 4.924 4.875 2 4.806 4.857 2 4.959 4.806 3 4.820 4.771 3 4.908 4.996 4 4.813 4.869 4 4.857 4.954 5 4.964 4.954 5 4.813 4.919 6 4.929 4.881 - - - 7 4.857 4.833 - - - 8 4.771 4.898 - - - 9 4.845 4.934 - - - 10 4.954 4.732 - - -

mean 4.873 4.901 SS 0.034 -

yx − - 0.028

σ̂ 0.190 0.190 0.3σ̂ 0.057 0.057

SS ≤0.3σ̂ PASS - yx − ≤0.3σ̂ - PASS

Note: * Compendium of method for the microbiological examination of foods (APHA), 2001 Chapter 20, p.209-213

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Table 4 Technical information in relation to the methods

Accredited Laboratory number

Yes 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 30 31 32 33 34 35 36 37 38 39

No 1 13 29 43

Reference Laboratory number

AOAC 1 2 3 4 5 7 8 9 10 13 15 17 18 20 23 24 26 27 28 29 30 31 33 34 36 38 39

APHA 22 25 35 FDA-BAM 11 14 16 19 21 32 37 42 ISO 21527-1 6 43 ISO 21527-2 12 40 41

Selective plating Laboratory number

Dichloran 18% glycerol agar 16 19 21 22 25 32 35 40 41 42 Dichloran Rose Bengal Chloramphenicol Agar 6 12 37 43 PCA + Chloramphenical 11 14 Petrifilm 1 2 3 4 5 7 8 9 10 13 15 17 18 26 28

30 33 34 38 39 Simplate for Yeast and Mold Color Indicator 20 23 24 27 29 31 36

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COMMITTEE

Technical consultants 1. Mrs. Nitaya Pirapatrungsuriya Vice President, Department of Laboratory Services 2. Mrs. Prachern Nakpan Manager, Division of Microbiological Laboratory

Statistician consultant

1. Chutima Waisarayutt, Ph.D. Department of Agro-Industrial Technology Faculty of Agro-Industry, Kasetsart University

Operators 1. Mrs. Phattraphorn Ketkaen Manager, Proficiency Testing Services Division

(Statistician) 2. Miss Tanaporn Borisut Scientist, Division of Proficiency Testing Services (Coordinator) 3. Miss Thatsanee Kansan Scientist, Division of Proficiency Testing Services

Proficiency testing provider accreditation ISO/IEC 17043: BLA-DSS Acc. No. PTP-0002 Approved by (Mrs. Nitaya Pirapatrungsuriya) Technical Manager 25 June 2014