Until 1946 there was a legal plate count standard of 100,000/ml for pasteurized milk in England and Wales. It had for long been realized that if a raw milk had more than 100,000 thermoduric organisms/ml then it was impossible to produce a pasteurized milk with a plate count of less than 100,000/ml, so that the test had fallen into disrepute. It was accordingly replaced by an incubated méthylène blue test (Table 13). This is controlled mainly by acid-producing types growing at atmospheric temperatures, and is a sound test for keeping quality. However, a high count of non-thermoduric types in the raw milk and/or a high count of biochemically sluggish thermo-duric types could produce a taint in milk without appreciably affecting keeping quality or the méthylène blue test on the pasteurized milk. Thermo-duric organisms are defined as those surviving pasteurization and in milk consist mainly of micrococci, Microbacterium, Corynebacterium, Strepto-coccus faecalis and Str. thermophilus. Spores survive but the vegetative cells of spore-bearing organisms are usually killed.
Scotland and Northern Ireland still retain the coliform test, which is excellent as an index of post-pasteurization contamination and thoroughly justifiable as a test and is equitable to the processor. However, the incubated
78 J. G. DAVIS
TABLE 13. Statutory bacteriological tests and standards for milk in England and Wales144
Product Test Standard
Raw milk (producers and
méthylène blue test responds to coliforms in pasteurized milk. Coliforms should be absent in 1 ml in pasteurized milk as sold. The common defects and their causes are summarized in Table 14 and bacteriological standards are suggested in Table 15.
TABLE 14. Pasteurized milk
Practically all milk used in manufacture to-day receives heat-treatment sufficient to kill at least 99 % of the raw milk flora. There is now an increasing tendency to use more drastic heat-treatment for milk for all purposes, e.g., 90°C for 15 sec for milk powder and cream for butter, 140°C for ^ 2 sec for sterilized milk, and even cheese milk is often now receiving the full legal
pasteurization (71 °C for 15 sec) instead of a simple "flashing" at about 68°C because of the danger of toxigenic staphylococci proliferating in the milk.
The bacterial count of the final product is therefore largely a measure of the sanitary condition of the equipment used and the efficiency of processing, particularly the speed of cooling, the prevention of after-contamination and the temperature of storage.
Pasteurization does not kill thermoduric bacteria or spores derived either from the raw milk or the equipment. All examinations and control measures in respect of these should, therefore, be applied both to the raw milk (p. 59) and the equipment (p. 60), although checks on the pasteurized milk may be made as required.
TABLE 15. Suggested bacteriological standards for pasteurized milk as sold (including homogenized milk)
Satisfactory Doubtful Unsatisfactory
Total count < 10,000 10,000-100,000 > 100,000 Non-thermoduric count < 1,000 1,000-10,000 > 10,000 Presumptive coli Absent in 1 ml Absent in 0-1 ml Present in 0-1 ml
Present In 1 ml
B. cereus spores <1 1-10 >10
Microbiological tests on pasteurized milk are most usefully directed to detect post-pasteurization contamination. Indeed by sampling at appropriate places in the equipment and making total counts and/or, after incubation for 24 hr at 20°C, dye reduction and/or presumptive coli tests it is possible to obtain a reliable assessment of the efficiency of cleansing (cleaning and
"sterilizing") the equipment so that swab and rinse tests (p. 60) become un-necessary. A simple and most useful method is to take the 1st, 50th, 100th etc., bottle or carton off the filler, hold a 10 ml sample for 24 hr at 20°C and then make a resazurin test at 37°C. Dirty equipment will be revealed by marked changes in resazurin in 30-60 min in the early bottle samples and this constitutes a very effective demonstration to the cleaning staff (see also p. 162).
Simple demonstration tests such as this have a far greater educational value than masses of results of more elaborate tests.
Although pasteurization properly carried out, and with no post-pasteur-ization contamination, always improves the keeping quality of milk as well as making it safe, it seems to be impossible to predict with any accuracy the increase in keeping quality, and there is a poor correlation between the temperature and time of pasteurization, the count of the pasteurized milk
80 J. G. DAVIS
and its keeping quality. With good hygienic control and a raw milk low in thermoduric organisms, pasteurized milk should keep for at least 1 day in hot weather (25°C) and 7 days at 5°C.
For experimental work a laboratory HT-ST pasteurizer can be used,294-5 but for nearly all purposes a laboratory holder method (63°C for 30 min) is satisfactory and simpler to operate.296
E. The Phosphatase Test
This test depends on the fact that the alkaline phosphatase of milk is almost completely destroyed by pasteurization, and is one of the most useful ever devised for the dairy industry. The original Kay-Graham test involving the estimation of phenol was subject to certain disadvantages297 and in the United Kingdom has been replaced by the Aschaffenburg-Mullen tech-nique80»144 using/7-nitrophenol, which is less subject to sources of error. In the U.S.A. modifications of the original phenol method are still in use. The phosph-atase enzyme is destroyed by a heat-treatment slightly more severe than that required to kill tubercle bacilli, so that a negative result indicates satisfactory pasteurization and a safe milk if not recontaminated, i.e. heating at a minimum temperature of 71°C for at least 15 sec or the equivalent. The test detects under-pasteurization and addition of as little as 0-1-0*2% raw milk. An automated form of the test has been developed.298
The phosphatase test does not detect post-pasteurization contamination and it does not measure keeping quality. It simply detects under-pasteuriza-tion of the milk or product. Reactivaunder-pasteuriza-tion of phosphatase may occur in milk heated above 82°C and in cream above 74°C.2"-302 Magnesium ions, /?-glycerophosphate and jß-lactoglobulin and other substances can act as factors in this as yet not understood phenomenon.303
F. "Bitty Cream"
Normally, spores are of little consequence in the dairy industry but there is a type of aerobic spore-former {Bacillus) which can occasionally cause havoc of epidemic proportions.
B. cereus and B. mycoides (a variant) produce lecithinase and by disrupting the membrane of the fat globule in milk can cause "bitty cream" (lumps of fat on the surface) and by rennet production can also cause "sweet curdling"
(non-acid coagulation). Pasteurization kills all non-thermoduric vegetative cells but not spores, consequently any spores of these organisms will survive.
If the temperature of the milk subsequently rises to 20°C or over (as it does in heat waves in the home) these spores can germinate and multiply rapidly and so produce this fault. There is no practicable cure, and the only known
preventive method is to hold the milk cold (below 10°C).304»305 Fortunately, the numbers of these spores in milk are usually very small306» 307 but an epidemic contamination could jeopardize all pasteurized milk. Aspects of techniques have been reported.308» 309