collaborative stdz of iso 26323|idf 213 on …

collaborative study of iso 26323|idf 213 on determination of the acidification activity of dairy cultures by continuous ph measurement (cph)

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Bulletin of the International Dairy Federation

Collaborative study of ISO 26323|IDF 213 on determination of the acidification activity of dairy cultures by continuous pH measurement (CpH)

4672013

Bulletin of the international Dairy feDeration 467/2013


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Collaborative study of ISO 26323|IDF 213 on determination of the acidification activity of dairy cultures by continuous pH measurement (CpH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Collaborative study of ISO 26323|IDF 213 on determination

of the acidification activity of dairy cultures by continuous pH

measurement (CpH)

Bulletin of the International Dairy Federation 467/2013Price: 12 Euro

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ISSN 0250-5118


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Recognizing the need for a standard method for measuring acidification activity of lactic acid bacteria used in milk fermentation, IDF and ISO developed the international standardized method ISO 26323|IDF 213:2008 - Milk products – Determination of the acidification activity of dairy cultures by continuous pH measurement (CpH). An international collaborative study was carried out on the basis of commercially available acidifying starter cultures. This report outlines the results of this study and provides the precision data for the international standard method. The dairy world and IDF thank Lasse Vigel Jørgensen (DK) and Rob Crawford (NZ), and the scientists who collaborated in this work (the laboratories are cited in the text) for their efforts in bringing the task to a satisfactory conclusion.

The development of the method was undertaken by the former Joint IDF/ISO action team on Lactic Acid Bacteria and Starters of the former Standing Committee on Microbiological Methods of Analysis (SCMMA), now replaced by the Standing Committee on Analytical Methods for Dairy Microorganisms.

Nico van Belzen, PhD Director General International Dairy Federation Brussels, August 2013


F O R E W O R D


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SUMMARy

An international collaborative study of ISO 26323|IDF213:2008, Milk products – Determination of the acidification activity of dairy cultures by continuous pH measurement (CpH), involving 10 laboratories located in 5 countries, was carried out on the basis of commercially available acidifying starter cultures according to ISO 5725-1 [3] and ISO 5725-2 [4]. The samples were prepared in double blind and analysed in duplicate.

The “IDF-CpH”-Ring trial was organized by Chr. Hansen A/S, Denmark. The method including all relevant instructions was submitted to all participating partners. After collection and preparation of the resulting data, statistical analyses were performed in close co-operation with the former IDF/ISO Joint Action team (JAT) on Statistics and sampling.

1. INTRODUCTION

For several years there has been a need from the dairy sector for an analytical method that is able to measure acidification activity of lactic acid bacteria used to ferment milk to produce dairy products. As a result of that IDF initiated a work item in the 1990’s to develop a procedure for a standard method for determination of acidifying activity of dairy cultures. The experts involved could not agree on a standard method. As a result a guideline was described with a high degree of variability of the different parameters important for the precision of the method [1], for example dry matter content and heat treatment of the milk used, sample preparation etc.

As a result of the need for a standard method for measuring acidification activity, a draft for a standard

method was proposed to IDF/ISO as a new work item. The work to develop the method was part of the former JAT Lactic Acid Bacteria and Starters, with the former Standing Committee on Microbiological Methods of Analysis (SCMMA).

2. MATERIALS AND METHODS

2.1. Participating laboratories:

The study included 10 laboratories (A-J) with varying degrees of experience with the method. The laboratories A-H trained in using the method over several years. This is in contrast to Laboratory I and J that have years of experience in measuring acidification activity but little experience with the specific parameters of ISO 26323|IDF213 [5]. In three of the laboratories (A, B and E) two different technicians did the whole dataset independently of each other. In six laboratories (C, F, G, H, I and J) one data set was generated by one technician. In laboratory D the analytical work was split between two technicians for practical reasons.

2.2. Samples:

The samples used in this study were commercially produced starter cultures normally used to produce dairy products around the world. The culture had been frozen by direct submission into liquid nitrogen in small pellets. These pellets were distributed into glass vials containing approximately 20 g. Ten vials of each bacterial culture were used for homogeneity testing in laboratory D. The sample vials were stored at


Lasse Vigel Jørgensen and Rob Crawford 1. Christian Hansen A/S, Boge Allé 10-12, DK-2970 Horsholm, Denmark

2. Fonterra Co-operative Group Limited, PO Box 459, Hamilton, New Zealand


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-50°C and shipped to the participating laboratories with dry ice to maintain the activity of the samples.

The bacterial cultures used in this study were:

- Sample 1: A mesophilic O-starter culture of Lactococcus lactis strains (O-940), - Sample 2: A mesophilic LD-starter culture of Lactococcus lactis, Leuconostoc mesenteriodes and

Lactococcus lactis subsp. Lactis (biovar.) diacetylactis strains. (CHN-11) and - Sample 3: A thermophilic starter culture of Streptococcus thermophilus strains (TH-4).

The method used by all laboratories was ISO/DIS 26323|ÍDF 213 Milk products – Determination of the acidification activity of dairy cultures by continuous pH measurement (CpH) [5].

3. DISCUSSION AND RESULTS

For each analytical trial, the pH of the milk was measured after 15 min (pH-15min) as a reference point for the milk preparation, pH calibration and the starting point for the acidification.

Several acidification parameters were measured:

ta: The time it takes to start acidifying the standardized milk, i.e.. the time for pH to drop 0.08 pH-units from initial pH (after 15 min). ta is measured in minutes from the inoculation time, t = 0. If the software is collecting data every 4 min, Ta should be measured by interpolation.pHxh: The pH after x hours of acidification in the standardized milk at 30 °C, 37 °C, 40 °C or 43 °C This could be the pH after 4 h, 6 h, 12 h or 16 h. The actual time for the parameter depends on the characteristics of the starter culture.

ta is usually described as the lag phase of the acidification and should not be confused with the classical lag phase for bacterial growth as defined by Monod [2] The interpretation of the parameter pHxh is dependent on the dose response of the individual bacterial cultures. In the present study pH-4h and pH-6h were used to measure acidification activity, whereas pH-16h was more regarded as an end-point measurement of the acidification.

The statistical calculation included detection of Cochran and Grubbs outliers and calculation of repeatability standard deviation (sr) and reproducibility standard deviation (sr). The results obtained in laboratory J were removed from the dataset as the laboratory was unable to master the method during the study.

The results are given in Figures 1 – 12.


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Figure 1: Average result from the collaborative study of CpH (ISO26323|IDF213). Sample 1: mesophilic O-starter culture of Lactococcus lactis strains (O-940). Bars indicate standard deviation of all results. The average and standard deviation of all laboratories has been calculated without the results of Lab J.

Collaboratory study of CpH - ISO26323|IDF213Lactococcus lactis O-940 - ta

111 111107

105

96

125

116 114 112 110114

108

142

111

50

60

70

80

90

100

110

120

130

140

150

A1 A2 B1 B2 C1 D1a/b E1 E2 F1 G1 H1 I1 J1 Avr+/-Sd

A A B B C D E E F G H I J All Labs(-J)

Technician / Laboratory

min

Collaboratory study of CpH - ISO26323|IDF213Lactococcus lactis, O-940 - pH15min

6,59 6,57 6,58 6,566,53

6,47

6,536,56

6,49 6,50

6,57 6,59

6,25

6,54

6,00

6,10

6,20

6,30

6,40

6,50

6,60

6,70

6,80

6,90

7,00




pH


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Collaboratory study of CpH - ISO26323|IDF213Lactococcus lactis O-940 - ta

111 111107

105

96

125

116 114 112 110114

108

142

111

50

60

70

80

90

100

110

120

130

140

150




min

Collaboratory study of CpH - ISO26323|IDF213Lactococcus lactis, O-940 - pH15min

6,59 6,57 6,58 6,566,53

6,47

6,536,56

6,49 6,50

6,57 6,59

6,25

6,54

6,00

6,10

6,20

6,30

6,40

6,50

6,60

6,70

6,80

6,90

7,00




pH



5

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Collaboratory study of CpH - ISO26323|IDF213Lactococcus lactis O-940 - pH6h

5,04 5,05 5,05 5,04

4,93

5,16

5,08 5,06 5,055,02

5,07 5,06

5,36

5,05

4,50

4,60

4,70

4,80

4,90

5,00

5,10

5,20

5,30

5,40

5,50




pH


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Figure 5: Average result from the collaborative study of CpH (ISO26323|IDF213). Sample 2:mesophilic LD-starter culture of Lactococcus lactis, Leuconostoc mesenteriodes and Lactococcus

Collaboratory study of CpH - ISO26323|IDF213Mesophilic LD-starter culture, CHN-11 - pH15min

6,60 6,606,63

6,58 6,606,56

6,656,62 6,60 6,58 6,60

6,58

6,33

6,60

6,00

6,10

6,20

6,30

6,40

6,50

6,60

6,70

6,80

6,90

7,00




pH


4,36 4,36 4,374,34 4,35 4,33

4,37 4,35

4,294,32

4,36

4,27

4,46

4,34

4,00

4,10

4,20

4,30

4,40

4,50

4,60

4,70

4,80

4,90

5,00




pH



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5


Figure 5: Average result from the collaborative study of CpH (ISO26323|IDF213). Sample 2:mesophilic LD-starter culture of Lactococcus lactis, Leuconostoc mesenteriodes and Lactococcus

Collaboratory study of CpH - ISO26323|IDF213Mesophilic LD-starter culture, CHN-11 - pH15min

6,60 6,606,63

6,58 6,606,56

6,656,62 6,60 6,58 6,60

6,58

6,33

6,60

6,00

6,10

6,20

6,30

6,40

6,50

6,60

6,70

6,80

6,90

7,00




pH


4,36 4,36 4,374,34 4,35 4,33

4,37 4,35

4,294,32

4,36

4,27

4,46

4,34

4,00

4,10

4,20

4,30

4,40

4,50

4,60

4,70

4,80

4,90

5,00




pH

Figure 5: Average result from the collaborative study of CpH (ISO26323|IDF213). Sample 2: mesophilic LD-starter culture of Lactococcus lactis, Leuconostoc mesenteriodes and Lactococcus lactis subsp. Lactis (biovar.) diacetylactis strains. (CHN-11). Bars indicate standard deviation of all results. The average and standard deviation of all laboratories has been calculated without the results of Lab J.

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lactis subsp. Lactis (biovar.) diacetylactis strains. (CHN-11). Bars indicate standard deviation of all results. The average and standard deviation of all laboratories has been calculated without the results of Lab J.


Collaboratory study of CpH - ISO26323|IDF213Mesophilic LD-starter culture, CHN-11 - ta

118 119116 117 117

119115 115 114

117122

113

172

117

80

90

100

110

120

130

140

150

160

170

180




min



7

7


Collaboratory study of CpH - ISO26323|IDF213Mesophilic LD-starter culture, CHN-11 - pH16h

4,51 4,514,53 4,52 4,52

4,494,52

4,494,44 4,45

4,51

4,45

4,60

4,50

4,00

4,10

4,20

4,30

4,40

4,50

4,60

4,70

4,80

4,90

5,00




pH


5,28 5,30 5,315,26

5,33 5,345,31

5,285,33

5,21

5,35 5,34

5,96

5,30

5,00

5,10

5,20

5,30

5,40

5,50

5,60

5,70

5,80

5,90

6,00




pH


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4,51 4,514,53 4,52 4,52

4,494,52

4,494,44 4,45

4,51

4,45

4,60

4,50

4,00

4,10

4,20

4,30

4,40

4,50

4,60

4,70

4,80

4,90

5,00




pH


5,28 5,30 5,315,26

5,33 5,345,31

5,285,33

5,21

5,35 5,34

5,96

5,30

5,00

5,10

5,20

5,30

5,40

5,50

5,60

5,70

5,80

5,90

6,00




pH



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-Figure 9: Average result from the collaborative study of CpH (ISO26323|IDF213). Sample 3:

thermophilic Starter culture of Streptococcus thermophilus strains (TH-4). Bars indicate standard deviation of all results. The average and standard deviation of all laboratories has been calculated without the results of Lab J.

Collaboratory study of CpH - ISO26323|IDF213Streptococcus thermophilus, TH-4 - pH15min

6,566,60 6,57 6,56 6,56

6,49

6,58 6,59

6,53 6,54 6,546,56

6,30

6,56

6,00

6,10

6,20

6,30

6,40

6,50

6,60

6,70

6,80

6,90

7,00




pH

Figure 9: Average result from the collaborative study of CpH (ISO26323|IDF213). Sample 3: thermophilic Starter culture of Streptococcus thermophilus strains (TH-4). Bars indicate standard deviation of all results. The average and standard deviation of all laboratories has been calculated without the results of Lab J.

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Figure 11: Average result from the collaborative study of CpH (ISO26323|IDF213). Sample 3: thermophilic Starter culture of Streptococcus thermophilus strains (TH-4). Bars indicate standard

Collaboratory study of CpH - ISO26323|IDF213Streptococcus thermophilus, TH-4 - ta

66 64 6562

65

77

6367 65

61

67 65

47

65

20

30

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min

Collaboratory study of CpH - ISO26323|IDF213Streptococcus thermophilus , TH-4 - pH4h

5,01 5,02 5,03 5,03 5,02

5,13

4,995,01

4,96 4,965,01

5,08

4,56

5,02

4,50

4,60

4,70

4,80

4,90

5,00

5,10

5,20

5,30

5,40

5,50




pH



9

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Figure 11: Average result from the collaborative study of CpH (ISO26323|IDF213). Sample 3: thermophilic Starter culture of Streptococcus thermophilus strains (TH-4). Bars indicate standard

Collaboratory study of CpH - ISO26323|IDF213Streptococcus thermophilus, TH-4 - ta

66 64 6562

65

77

6367 65

61

67 65

47

65

20

30

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50

60

70

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90

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110

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min


5,01 5,02 5,03 5,03 5,02

5,13

4,995,01

4,96 4,965,01

5,08

4,56

5,02

4,50

4,60

4,70

4,80

4,90

5,00

5,10

5,20

5,30

5,40

5,50




pH


10



4,20 4,204,23 4,24 4,22

4,154,18 4,17

4,114,16

4,21

4,16 4,16 4,19

4,00

4,10

4,20

4,30

4,40

4,50

4,60

4,70

4,80

4,90

5,00




pH



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4. CONCLUSION

The summary of the collaborative study is summarized in table A.1. Apart from the results of laboratory J very few outliers was detected by the Cochran and Grubbs tests.

The precision data for the collaborative study have been incorporated into the International Standard.

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Table A.1 — Results of the collaborative study

PARAMETER

Samples

Sample 1 Sample 2 Sample 3

ta pH 6h pH 16h ta pH 6h pH 16h ta pH 4h pH 16h

N° of laboratories 10 10 10 10 10 10 10 10 10

N° of samples 3 3 3 3 3 3 3 3 3

N° of participants 15 15 15 15 15 15 15 15 15

Mean Value 110.7 5.05 4.34 116.8 5.30 4.50 65.5 5.02 4.19

N° of accepted datasets 12 12 12 12 12 12 12 12 12

N° of results used to estimate r, R 48 47 47 48 47 47 44 48 48

Repeatability, standard deviation, sr

2.6 0.014 0.011 2.8 0.02 0.010 1.4 0.173 0.014

Repeatability value, r(=2.8 sr) 7.3 0.039 0.032 7.8 0.062 0.028 4.0 0.048 0.039

Reproducibility, standard deviation sR

7.5 0.055 0.032 4.3 0.047 0.034 2.3 0.050 0.040

Reproducibility value, R (=2.8 sR) 21.1 0.154 0.090 12.0 0.131 0.094 6.5 0.140 0.111

Table A.1 — Results of the collaborative study

Description of test samples used : Samples 1 A mesophilic O-starter culture of Lactococcus lactis strains

Samples 2 A mesophilic LD-starter culture of Lactococcus lactis, Leuconostoc mesenteriodes and Lactococcus lactis subsp. Lactis (biovar.) diacetylactis strains.

Samples 3 A thermophilic Starter culture of Streptococcus thermophilus strains.


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5. REFERENCES

[1] IDF Guideline - Determination of acidifying activity of dairy cultures - by Group E104 - Lactic acid bacteria and starters. Bulletin of the IDF 306/1995

[2] The Growth Of Bacterial Cultures, Jacques Monod, Pasteur Institute, Paris, France, Annu. Rev. Microbiol. 1949.3:371-394).

[3] ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results. Part 1: General principles and definitions.

[4] ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results. Part 2: A basic method for the determination of repeatability and reproducibility of a standard measurement method.

[5] ISO 26323|IDF 213:2009 – Milk products - Determination of the acidification activity of dairy cultures by continuous pH measurement (CpH)


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COLLABORATIVE STUDy OF ISO 26323|IDF 213 ON DETERMINATION OF THE ACIDIFICATION ACTIVITy OF DAIRy CULTURES By CONTINUOUS PH MEASUREMENT (CPH)

Lasse Vigel Jørgensen and Rob Crawford

ABSTRACT

This report compiles the results of an international collaborative study conducted to establish precision figures for the determination of acidification activity of dairy cultures by continuous pH measurement with the method described in ISO 26323|IDF 213. The cultures used were: mesophilic O-starter culture of Lactococcus lactis strains, mesophilic LD-starter culture of Lactococcus lactis, Leuconostoc mesenteriodes and Lactococcus lactis subsp. Lactis (biovar.) diacetylactis strains and thermophilic starter culture of Streptococcus thermophilus strains. The summary of the collaborative study is included in the report and into the International Standard.

Keywords: Acidification activity, dairy cultures, interlaboratory study, pH measurement, repeatability

11 pp - English only

Bulletin of IDF N° 467/2013 – Price (electronic) 12 Euro – Date 2013


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