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ISO 26323:2009 — Determination of Acidification Activity of Dairy Cultures by Continuous pH Measurement (CpH)
A comprehensive technical guide to the ISO standard for measuring lactic acid bacteria activity in dairy starter cultures, jointly published with IDF 213:2009.
1. Introduction and Scope
ISO 26323:2009, jointly published with IDF (International Dairy Federation) as IDF 213:2009, specifies a standardized method for measuring the acidification activity of lactic acid bacteria (LAB) in dairy starter cultures through continuous pH measurement (CpH). The method is applicable to dairy starter cultures used in the production of fermented milk products, cheese, yogurt, and other cultured dairy foods.
Why Continuous pH Measurement? Traditional endpoint pH measurements only capture the final acidity after a fixed incubation period. Continuous pH measurement reveals the entire acidification kinetics — the lag phase, exponential acid production phase, and the approach to the final pH — providing far richer information about culture performance and consistency.
The standard specifies two types of standardized milk substrates: boiled milk with 9.5 % dry matter (B-milk 9.5) and autoclaved milk with 9.5 % dry matter (A-milk 9.5). B-milk 9.5 may retain residual enzyme activity that can affect certain cultures; in such cases, A-milk 9.5, where all enzymes have been thermally inactivated, is used instead.
The method is applicable to both frozen and freeze-dried (lyophilized) starter culture formats, making it versatile across different commercial product types. The standard defines key acidification parameters including ta (time to initial pH drop), pHt (pH after a specified time), and tpH x (time to reach a target pH).
2. Principle and Key Parameters
A specified quantity of starter culture is diluted and inoculated into standardized milk, then incubated at a constant temperature (30 °C, 37 °C, 40 °C, or 43 °C, depending on the culture type). The acidification activity is monitored by continuous pH measurement using a pH electrode and data logger. From the fermentation curve, several parameters can be extracted:
| Parameter | Symbol | Description | Typical Use Case |
|---|---|---|---|
| Initial pH | pH15min | pH measured 15 minutes after inoculation, ensuring electrode and milk have reached equilibrium | Baseline quality check |
| Time to acidify | ta | Time (minutes) for pH to drop 0.08 pH units from the initial pH after 15 min | Measure of early lag phase activity |
| pH at time t | pHt | pH after a specified time (e.g., pH4h, pH6h, pH12h, pH16h) | Mid-to-late fermentation activity |
| Time to target pH | tpH x | Time required to reach a specified pH (e.g., pH 4.50) | Process control for fermentation endpoint |
Engineering Insight: The ta parameter is particularly sensitive to the physiological state of the culture upon inoculation. A prolonged ta (>120 min for mesophilic cultures) can indicate cold shock from improper thawing, bacteriophage contamination, or antibiotic residues in the milk substrate. Monitoring ta alongside pH6h provides a powerful diagnostic pair for troubleshooting culture performance issues.
3. Reagents and Equipment
3.1 Standardized Milk Substrate
The quality of the milk substrate is critical for reproducible results. The standard specifies medium-heat, low-fat, spray-dried milk powder with the following composition:
| Component | Mass Fraction (%) |
|---|---|
| Milk protein | 34–38 |
| Lactose | 48–56 |
| Milk fat | < 1.25 |
| Ash | 7–9 |
| Moisture | < 4 |
| Titratable acidity (as lactic acid) | < 0.15 |
The reconstituted milk (QC-Milk 9.5) is formulated to a 9.5 % ± 0.2 % dry matter content using 10.9–11.2 kg of milk powder per 100.0 kg of water. The dry matter level is similar to bottled sterilized milk, providing a consistent and reproducible fermentation environment.
3.2 Essential Equipment
The following equipment is specified for the CpH method:
- Analytical balances — capable of 0.01 g and 1 mg precision
- Autoclave — capable of operating at 99 °C ± 1 °C (B-milk) and 115 °C ± 1 °C (A-milk)
- Temperature-controlled water baths — capable of maintaining 30.0 °C, 37.0 °C, 40.0 °C, and 43.0 °C ± 0.2 °C
- pH electrodes — suitable for continuous immersion; e.g., Mettler Toledo 405-DPAS-SC-K8S/150 or equivalent
- Cylindrical bottles — 250 ml capacity, 16.5 cm height, 5.5 cm internal diameter
- Temperature probes — calibrated accuracy of ± 0.1 °C
- Data logger — equipped with pH and temperature channels, connected to data acquisition software
Critical Note on Electrode Maintenance: The standard dedicates significant attention to pH electrode cleaning and calibration. Between runs, electrodes must be rinsed, cleaned with ethanol to remove fat residues, immersed in pepsin/HCl electrode cleaner for at least 15 minutes, and stabilized in 3 mol/l KCl solution for 30 minutes. Neglecting electrode maintenance is the single most common cause of poor reproducibility in CpH measurements. The calibration slope must be ≥ 93 % with an intercept between −30 mV and +30 mV.
4. Procedure for Frozen and Freeze-Dried Cultures
4.1 Frozen Cultures
For frozen cultures, the sample (10 g ± 4 g for single-component products) is thawed in a water bath at 21 °C until just thawed (< 20 minutes), taking care that the temperature does not exceed 5 °C. The inoculation uses a two-stage weighing procedure:
- First weighing (mI1): 1.5–2.5 g of thawed culture is weighed into a milk bottle for dilution
- Second weighing (mI2): After mixing, 0.8–2.9 g (depending on target inoculation %) is transferred into the preheated activity milk bottle
The inoculation percentage wI is calculated as: wI = (mI1/m1) × (mI2/m2) × 100, where m1 and m2 are the total masses at each weighing stage.
4.2 Freeze-Dried Cultures
Freeze-dried cultures are acclimatized to room temperature for 15 minutes before opening. A three-stage weighing procedure is used, with the target inoculation calculated from the manufacturer’s recommended dosage. The third weighing (mI3) is transferred into the warm activity milk bottle using a pipette.
The temperature programme for heat-treating the milk substrates is as follows:
| Parameter | B-milk 9.5 (Boiled) | A-milk 9.5 (Autoclaved) |
|---|---|---|
| Holding temperature | 99 °C ± 1 °C | 115 °C ± 1 °C |
| Holding time | 30 min ± 1 min | 15 min ± 1 min |
| Cooling | 99 °C → 40 °C in < 40 min | 115 °C → 40 °C in < 40 min |
| Storage | < 7 °C for 16 h min to 12 d max | < 7 °C for 16 h min to 12 d max |
| Enzyme activity | Residual proteases possible | Fully inactivated |
Quality Assurance: Always run a control culture with known activity in parallel with test samples using the same time, temperature, and water bath conditions. The temperature profile measured by a calibrated probe in an uninoculated control bottle must remain within ± 0.5 °C of the target temperature for the run to be approved.
5. Precision and Interlaboratory Validation
An international collaborative study (CpH ring trial) involving 10 laboratories across 5 countries was conducted according to ISO 5725-1 and ISO 5725-2 to determine the precision of the CpH method. Three commercial dairy starter cultures were tested:
- Sample 1: A mesophilic O-starter culture of Lactococcus lactis strains
- Sample 2: A mesophilic LD-starter culture of Lactococcus lactis, Leuconostoc mesenteroides, and Lactococcus lactis subsp. lactis biovar. diacetylactis strains
- Sample 3: A thermophilic starter culture of Streptococcus thermophilus strains
| Parameter | Sample 1 (r / R) | Sample 2 (r / R) | Sample 3 (r / R) |
|---|---|---|---|
| ta (min) | 7.3 / 21.1 | 7.8 / 12.0 | 4.0 / 6.5 |
| pH6h (Sample 1, 2) / pH4h (Sample 3) | 0.039 / 0.154 | 0.062 / 0.131 | 0.484 / 0.140 |
| pH16h | 0.032 / 0.090 | 0.028 / 0.094 | 0.039 / 0.111 |
Here, r is the repeatability limit (within-laboratory) and R is the reproducibility limit (between-laboratories). The data show that the CpH method achieves good precision, particularly for pH parameters, with reproducibility standard deviations typically below 0.06 pH units for most measurements.
Engineering Insight: The higher reproducibility variability for ta (R = 21.1 min for Sample 1) compared to pH parameters reflects the inherent sensitivity of the lag phase to subtle differences in substrate preparation and temperature equilibration between laboratories. For routine quality control, pH6h or pH16h provide more robust inter-laboratory comparability. If ta is critical for your application (e.g., fast-fermenting thermophilic cultures), invest in strict temperature control (±0.1 °C) and standardized milk powder sourcing.
6. Engineering Design Insights and Best Practices
Implementing a reliable CpH testing program requires attention to several engineering aspects beyond the basic procedural steps:
6.1 Water Quality
The water used for milk reconstitution must have a bacterial count below 50 cells/ml and conductivity below 5 µS/cm. Chlorinated water must be neutralized before use. These requirements are often overlooked but are critical for eliminating background interference in the acidification measurement.
6.2 Electrorode Management
Implement a strict electrode rotation and maintenance schedule. The standard recommends:
- Daily calibration renewal
- Weekly KCl solution replacement for storage
- Regular protein/fat cleaning between each fermentation run
- Heat decontamination at 99 °C for 30 min if bacteriophage contamination is suspected
6.3 Data Logging Configuration
If the data logger collects readings every 15 seconds, the ta parameter (time for 0.08 pH drop from pH15min) can be determined with a resolution of 0.25 minutes. Configure the logging software to automatically calculate and report ta, pHt, and tpH x parameters to reduce manual data processing errors.
6.4 Temperature Control
The water bath temperature must be controlled to ±0.2 °C. For large-scale testing programs, consider using circulating water baths with external temperature sensors rather than static baths, as the heat capacity of multiple bottles can cause transient temperature drops upon insertion.
Key Takeaway: ISO 26323:2009 provides a robust, internationally validated method for characterizing starter culture acidification activity. When properly implemented with attention to electrode maintenance, temperature control, and milk substrate quality, the CpH method delivers the precision needed for both quality control release testing and research and development optimization of fermented dairy products.
7. Frequently Asked Questions
Q1: What is the difference between B-milk 9.5 and A-milk 9.5, and when should I use each?
B-milk 9.5 is prepared by boiling (99 °C for 30 min) and may retain residual enzyme activity. It is suitable for most robust starter cultures. A-milk 9.5 is autoclaved (115 °C for 15 min), fully inactivating all enzymes. Use A-milk 9.5 when testing cultures that show poor repeatability with B-milk 9.5, particularly if residual protease activity is suspected to interfere with the acidification measurement.
Q2: How should I handle cultures with very slow or very fast acidification activity?
For slow cultures, extend the monitoring period to 16–24 hours and focus on pH16h or tpH 4.50 parameters. For very fast cultures (e.g., concentrated thermophilic cultures), reduce the inoculation percentage or use a higher dilution factor in the first weighing stage to bring the acidification curve within the measurable range.
Q3: My pH readings show drift during the measurement. What could be wrong?
pH drift during CpH measurements is most commonly caused by (a) fouling of the pH electrode diaphragm with milk protein, (b) inadequate KCl solution in the electrode reference junction, or (c) temperature instability in the water bath. Check the calibration slope (≥93 %) and intercept (−30 to +30 mV) before each run, and clean the electrode using the pepsin/HCl cleaner for at least 15 minutes between runs.
Q4: Can this method be used for non-dairy fermentation substrates?
The standard is specifically validated for milk-based substrates with 9.5 % dry matter. However, the principle of continuous pH measurement can be adapted to other substrates with appropriate validation. Note that the precision data (repeatability and reproducibility limits) apply only to the specified dairy matrices and culture types tested in the interlaboratory study.
