Process and Quality Control Procedures

6 Culture and Rennet Activity Tests

1, Culture Activity


This simple test is useful to ensure that cheese cultures have adequate activity before inoculating the cheese vat. For most cheese, a general rule of thumb is that the activity and amount of inoculum should be sufficient to produce a titratable acidity of about 0.34% lactic acid in 10% reconstituted skim milk after 4 h of incubation at 37ºC. The test is also useful to compare types of cultures prepared under different conditions. For these purposes a pH versus time chart is quite useful (See Figure 10.1). Another application is to check sensitivity of the culture to bacteriophage in the plant.

And, a variation of the culture activity test is  the Pearce test, follows the pH while the temperature is varied over time to mimic the temperature profile of making a particular cheese. This allows the cheese maker to determine the likely suitability of a culture for a particular cheese variety.


  1. Mix 10 g of low-heat, antibiotic-free skim-milk powder in 90 ml of distilled water in a 100 ml Erlenmeyer flask.
  2. Sterilize at 15 lb pressure (1.05 kPa) for 10 min.
  3. Cool to 37ºC.
  4. Inoculate with 3.0 ml starter or other amount as appropriate. Rinse pipette twice by drawing the sterile milk into it.
  5. Incubate at 37ºC for at least 4 h. Longer if desired for pH versus time profile.
  6. Check pH at 30 min. intervals.
  7. Titrate 17.6 ml with N/10 sodium hydroxide (NaOH) using 1 ml phenolphthalein. Divide the required ml of NaOH by 2 to obtain titratable acidity in units of percent lactic acid.
  8. Record starter activity as follows:

Active               >0.34%

Slow                 0.26-0.30%

Culture Activity Test: Example


Culture: Lactococcus lactis subsp. lactis

     Lactococcus lactis subsp. cremoris

Temperature: 37ºC

Inoculum: 2% mother culture prepared with 10% reconstituted skim milk powder

Test media 1: 10% skim milk powder, low heat, antibiotic free.

Test media 2: Same as one with 1% cheese whey.


Titratable acidity after 4 hours:

Treatment 1: 0.34%

Treatment 2: 0.25%

Table 6.1: Culture Activity Test. pH of test media versus time.

Time (h) 0 1 2 3 4 5 6 7 8 9
pH of Skim Powder 6.62 6.59 6.5 6.4 6.15 5.74 5.39 5.08 4.92 4.87
pH of Skim Powder with Whey 6.61 6.57 6.5 6.42 6.35 6.31 6.3 6.3 6.29 6.29


  1. Test media 1 shows normal growth. 0.34% acidity after 4 h with a 2% inoculum is adequate for most types of cheese. pH versus time plot is typical, reaching pH 5.2 between 6 and 7 hours.
  2. Test media 2 shows inadequate acid development indicating the probable presence of bacteriophage in the cheese plant.

[See Figure 10.1]


2. Rennet activity

Coagulation Time versus Setting Time

Rennet is traditionally described in the industry as single, double, or triple strength. Single strength is considered to be that concentration where 200 ml is sufficient to set 1,000 Kg of milk in 30 – 40 min at 30 – 32ºC. In terms of International Milk Clotting Units (IMCU), single strength rennet has about 250 IMCU per ml. Setting time is the point where the curd will break cleanly and exude clear whey. Coagulation time is the point where flecks of curd first appear on a spatula or slide dipped into the milk. Coagulation time is about half of setting time, so typically, coagulation using single strength rennet requires 15 – 20 minutes followed by setting at 30 – 40 minutes. The following simple test can be used to check coagulation time, which can be measured much more accurately than setting time. The test uses skim milk because the presence of fat globules makes it difficult to see the first sign of coagulation.

Measurement of Coagulation Time

  1. Prepare 200 ml samples of 10% reconstituted low heat skim milk powder in 250 ml beakers. Add 0.02% calcium chloride dehydrate (40 mg per 200 ml).
  2. Temper to 32ºC in a water bath.
  3. Add 1.0 ml of 5% single strength rennet solution to each sample.
  4. Determine the clotting time by repeatedly dipping a clean spatula or glass slide into the milk. When coagulation has occurred flecks of curd will appear in the milk film on the slide.

Relative Milk-clotting Activity Test

A more rigorous test of coagulant activity is the “Relative Milk-Clotting Activity Test” (RMCAT) which measures the activity of rennet and other coagulants in “International Milk-Clotting Units” (IMCU). [1] The current proliferation of rennet products with widely varying activities makes it necessary to define commercial products in terms of IMCU rather than single, double, or triple strength. The amount of rennet required for most cheese making purposes is about 50 IMCU per litre.

  1. The method is described in International Dairy Federation. (1992). Standard 157 - Determination of Total Milk-Clotting Activity of Bovine Rennets. International Dairy Federation, Brussels.


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