A Summary of Titratable Acidity

A Summary of Titratable Acidity

http://drinc.ucdavis.edu/dairychem5_new.htm 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 onAlkaline Phosphatase MethodologyEnzymes 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 METHODOLOGYDick H. Kleyn, Ph.D.,Dept. of Food ScienceRutgers UniversityNew Brunswick, NJ 08903