A Summary of Titratable Acidity

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1. There are two fundamentally different methods of expressing acidity: (a) titratable
acidity expressed as percent lactic acid, and (b) hydrogen ion concentration or pH.
The former measures the total acidity but does not measure the strength of the
acids. The pH indicates the strength of the acid condition.
2. The true neutral point is at pH 7.0; pH values below 7.0 indicate an acid
reaction; pH values above 7.O indicate an alkaline reaction. One pH unit means a
tenfold difference in strength; for example, a pH 5.5 indicates an acidity that is ten
times as great as pH 6.5.
3. It is the pH that determines such processes as curdling of milk the action of
enzymes, the growth of bacteria, the color of indicators, taste, etc. Today pH is easy
to measure.
4. The customary practice of using a 9 cc. sample in titrating dairy products
involves a weight discrepancy. Since the acidity test is used for comparative
purposes, this discrepancy is not serious. However, when we compare different
products (i.e.. milk and cream, milk and condensed milk), this discrepancy must be
remembered.
5. When titrating dairy products to the pink color of the phenolphthalein
endpoint, we are titrating to about a pH 8.3 or 8.4, which is quite appreciably on the
alkaline side of the true neutral point. On the other hand titrating to A pH of 7.0 will
result in a different % acidity. Most "normal" acidities reported in the early literature
were determined with the phenolphthalein indicator. Thus, the true acidity is reported
higher than it actually is.
6. If we add distilled water to the measured sample of milk, a lower titratable
acidity value is obtained. This is due to the fact that there will be less precipitation of
tri-calcium phosphate.
7. The acidity of milk from individual cows ranges from 0.10 to 0.26 %. Herd milk
varies less in acidity because of commingling,, but occasionally herds are found
where the acidity of the fresh milk is 0.18 % and as high as 0.23 %.
8. The acidity of fresh milk is due to phosphates, casein and whey proteins,
citrates and carbon dioxide.
9. The acidity of milk may change during the lactation period but no definite
trend can be stated except that (a) the acidity of colostrum is high, and (b) the milk
towards the very end of the lactation period frequently has a lower acidity.
10. It has been impossible to increase the acidity of milk by feeding silage or
even by feeding such inorganic acids such as inorganic as sulphuric acid or
phosphoric acid.
11. Mastitis, even in mild or sub-clinical form, causes the acidity of the milk to be
lower. In rare cases mastitis causes a high acidity in the milk.
12. The acids produced by bacteria growing in milk are mainly lactic and acetic
acids.
13. The number of bacteria must increase to several millions per mL before there
is a measurable increase in acidity.

 14. The acidity test must be used with considerable discretion, if used at all, for

grading raw milk at the plant intake, because (a) fresh milk varies widely in acidity,
and (b) millions of bacteria are required to produce the first rise in acidity. This
bacterial growth can only occur if the farm milk was held un- refrigerated at 55 F or
more. for several hours. If this were the case the required recording thermometer
would show a refrigeration problem. This "high acidity" milk would also have show a
SPC in excess of 500,000 per mL.
15. The expected acidity of cream is frequently calculated from the acidity of the
milk and the fat content of the cream. This calculation does not hold unless the
acidity of the cream is determined by measuring g cc. of cream, adding 9 cc. of
water, and then titrating as usual. Failure to recognize this fact has led to
unjustifiable neutralization of sweet cream.
16. If the acidity of condensed milk products is determined for the purpose of
forming an opinion as to the raw material used, then the sample should be measured
as in the case of milk or skim milk. Enough distilled water should be added to the
measured sample to dilute the condensed product to the original concentration of the
milk.
17. The acidity of ice cream mixes is higher than the acidity of milk in the same
proportion that the serum solids content of the mix is higher than that of milk.

A Brief Review on
Alkaline Phosphatase Methodology
Enzymes are organic catalysts which occur naturally in most raw foods. When milk is
pasteurized most of the enzymes are inactivated or their activity is greatly
diminished. The first reliable enzymatic test for determining efficiency of
pasteurization was developed by Kay and Graham in England in 1933. It was based
upon the inactivation of alkaline phosphatase.
The phosphatase test is applied to dairy products to determine whether
pasteurization was done properly and also to detect the possible addition of raw milk
to pasteurized milk. The thermal resistance of alkaline phosphatase has been
considered to be greater than that of any nonsporeforming pathogens that might be
found in milk. However, the recent outbreaks of disease traced to Listeria
monocytogenes in pasteurized milk lead one to begin to question this conclusion.
Alkaline phosphatase is a monesterase that catalyzes the hydrolysis of monoesters.
Studies have shown that the amount of alkaline phosphatase in raw milk is variable.
The activity of phosphatase per unit of milk seems to be inversely correlated to milk
yield, reaching a minimum in 1 or 2 weeks after calving and rising gradually to a
maximum in about 25 weeks. Breed, feed of the cow, or fat content of the milk do
not appear to influence phosphatase activity. Alkaline phosphatase is associated with
the fat globule of milk, i.e., it is adsorbed to the fat globule membrane surface.
Phosphatase tests currently described in Standard Methods for the Examination of
Dairy Products are based on the principle that the alkaline phosphatase enzyme in
raw milk liberates phenol from a disodium phenyl phosphate substrate (Scharer
Method) or phenolphthalein from a phenolphthalein monophosphate substrate

(Rutgers Method) when tests are conducted at suitable temperature and pH. The
amount of phenol or phenolphthalein liberated from the substrate is proportional to
the activity of the enzyme. Phenol is measured calorimetrically after its reaction with
2,6 dichloroquinone-chloroimide (CQC) to form indophenol. Phenolphthalein is
detected by addition of sodium hydroxide.
While the Scharer rapid method is relatively simple and quick, it must be recognized
that it does possess some inherent weaknesses. There is a constant hazard of phenol
contamination from reagents, glassware, and stoppers. Reagents are unstable as is
the color formed by the reaction of phenol with the dye. Visual measurement of color
is sometimes difficult, particularly with borderline cases; and emulsification
frequently occurs during the extraction of the phenol with butanol.
Phenolphthalein monophosphate is a very stable substrate which is easily hydrolyzed
by alkaline phosphatase to yield free phenolphthalein. Our studies have revealed that
the use of this substrate provides greater sensitivity than disodium phenyl
phosphate. The high sensitivity is due to several factors, namely, the ease of color
comparison, the high rate of hydrolysis, the elimination of variations because of
specific color reaction and extraction, and the slight contribution of yellow color of
milk fat to the pink color of phenolphthalein.
It is necessary to run both positive and negative controls when conducting a
phosphatase procedure. A negative control is prepared by heating a product to 90C
for 1 minute followed by rapid cooling. Any color developing when a test is run on
the control indicates contamination of reagents or presence of interfering coloring
materials or both. A positive control is run as a check on the proper functioning of
reagents. It is conducted by adding 0.2 ml of fresh, raw mixed-herd milk to 100 ml
of raw milk which has been heated at 90C for 1 minute, followed by rapid cooling to
room temperature. One should obtain a positive result on this test.
Another control test should be run on samples which yield positive results in the
initial analysis. This test is conducted in order to distinguish residual alkaline
phosphatase from microbial alkaline phosphatase. Microbial phosphatases are
considerably more heat resistant than is alkaline milk phosphatase. Therefore, it is
possible to differentiate these enzymes by pasteurization of the sample in question
and retesting. If there is no significant difference in the results of the test, one then
concludes that the original positive result was due to microbial phosphatase.
Reactivated phosphatase sometimes occurs in high fat dairy products which have
been ultrapasteurized, such reactivation occurring quickly when samples are stored
at non-refrigerated (70-90F) temperatures. A test has been developed which
permits one to distinguish residual from reactivated alkaline phosphatase.
Alkaline phosphatase methodology is applicable to cheese. However, consideration
must be given to the possibility of obtaining false-positive tests due to the possible
presence of mold in the cheese. In the early 1940's, Scharer reported as follows:
"Our recent work has indicated that yeast and some molds (a culture of Oidium lactis
and Penicillium notatum) which grown on cheese under certain conditions will
produce appreciable amounts of phosphatase, but that if the mold growth is removed
before the cheese sample is prepared for testing purpose, no difficulty or false
positive is encountered." Thus, sampling of cheese is a very important consideration,
i.e., one must be certain that no mold is evident. In addition, it is highly

recommended that cheese be sampled before the addition of condiments such as

peppers or spices as these materials may also be responsible for false positive tests.
Ideally, cheese samples should be placed in clean containers, refrigerated, and tested
within 36 hours in order to be certain that no development of microbial phosphatase
has occurred. Finally, if cheese is not properly stored in the marketplace there is a
possibility that microbial growth may occur which could result in false positive results
for the alkaline phosphatase test. Thus, sampling of cheese for phosphatase analysis
should be conducted before cheese enters marketing channels.
ALKALINE PHOSPHATASE METHODOLOGY
Dick H. Kleyn, Ph.D.,
Dept. of Food Science
Rutgers University
New Brunswick, NJ 08903