BACKGROUND OF THE INVENTION The present invention relates generally to compositions for inhibiting bacterial degradation of milk, and more particularly to a composition including an indicator which changes color in the event such degradation occurs. It is common practice in the dairy industry to send samples of various dairy products to centralized laboratories for testing. Typical measurements include the content of fat, protein, lactose, total solids, and added water. The samples may be further subjected to direct microscopic somatic cell count (DMSCC) which measures the content of bovine epithelial cells and serves as an indication of udder infection.

The test samples can be in transit for one or more days between the dairy farm and the central testing laboratory, and even when refrigerated, are subject to various forms of bacterial degradation.

The most common form of bacterial degradation encountered arises from the growth of lactic acid bacteria, in particular streptococci and lactobacilli. These bacteria are present in raw milk and utilize lactose to produce lactic acid. Their ability to produce acid and proliferate at low pH favors their survival over other common organisms in milk and other dairy products. As the pH of milk is lowered, casein, the principal milk protein having an isoelectric point at a pH of 4.6, begins to precipitate. The precipi¬ tation is first noticeable as a very fine graininess at a pH of about 5.2 (fresh milk has a pH of 6.7). The milk forms a solid gel at a pH of about 4.6-4.7.

Another form of bacterial degradation is caused by proteolytic microorganisms which enzymatically denature milk protein. Proteolysis is accompanied by acid production when caused by certain species of Micrococcus and (to a lesser extent) by species of

Bacillus and other groups. Such proteolytic degradation will predominate under conditions which are unfavorable to the growth of lactic acid bacteria.

The bacterial degradation of a dairy sample prior to testing is undesirable for two primary reasons. First, such degradation can directly affect the composi¬ tion of the sample. For example, lactic acid conversion can reduce the lactose measurement while protein precipitation can lower the measured protein content. Extensive growth of proteolytic and lipolytic bacteria can also lower protein and fat measurements by denatur¬ ing these components.

Second, and more importantly, the presence of ^• even small amounts of precipitated protein can foul the test equipment. Such test equipment is typically automated and relies on the transfer of fluid milk samples in narrow diameter capillary tubing. Precip¬ itated protein will quickly clog such tubing, requiring that the test equipment be shut down while the passage- ways are cleared. Moreover, since the test equipment usually measures light absorption through the sample, precipitated protein will increase the observed light absorption leading to a false reading.

To overcome the problems associated with bacterial degradation of the milk samples, preservative (anti-bacterial) compositions are usually added to the samples at the dairy. A number of such preservatives are commonly in use, including potassium dichromate, boric acid, and 2-bromo-2-nitropropane-l,3-diol. While generally effective, individual milk samples can still" suffer from bacterial degradation, particularly if they have been held for extended periods without refrigera-

tion. Under such circumstances, it is very easy for the central testing laboratory to allow degraded samples into their test instrumentation. It is very difficult to detect the early stages of degradation when only a small amount of the protein has precipitated. Such in¬ cipient precipitation, however, can adversely effect the measurements taken and foul the test instrumentation as described above.

For the above reasons, it would be desirable to provide a screening technique which would allow the personnel at the central test facility to easily determine when individual milk samples have been degraded and are unsuitable for testing.

SUMMARY OF THE INVENTION Novel preservative compositions having pH-sensitive color indicators incorporated therein are useful for indicating the degree of bacterial growth that has occurred in milk samples intended for chemical analysis. The color indicators are sensitive to pH changes in the range from about 6.6 to 4.6.

DESCRIPTION OF THE SPECIFIC EMBODIMENT

Bacterial growth in samples of milk and other dairy products can directly affect their suitability for testing. Such samples become unsuitable for testing when bacteria consume the constituents to be analyzed, such as lactose and protein, and when they act on the milk protein to change its degree of solubi¬ lity. The majority of microorganisms capable of decomposing milk exhibit some level of acid production. The present invention relies on the detection of incipient acid production as an indication of when a particular milk sample has undergone unacceptable degradation. The color indicator is combined with a preservative compound for simultaneous addition to the

milk sample while it is still fresh, usually at the dairy.

The preservative composition having a color indicator of the present invention is useful with all liquid (non-cultured) milk products which would be expected to be free of lactic acid. Such products are typified by raw whole milk; homogenized milk, including whole milk, partly-skimmed milk, and skimmed milk; cream and the like. The composition of the present invention will not be useful on those dairy products, such as cheese, cottage cheese, and yogurt, which have been cultured and in which lactic acid would naturally be present.

The present invention allows detection of bacterial degradation by any species capable of acid production in milk and milk products. As stated above, the most common organisms are streptococci and lactoba- cilli, which convert lactose to lactic acid. Certain species of Bacillus responsible for sweet-curd formation, however, also produce lactic acid in sufficient amounts to allow detection by the present invention. Other common microorganisms responsible for the degradation of milk and which produce detectable amounts of acid include certain strains of micrococci, microbacteria, pseudomonads and enteric bacteria. Some of these strains are also proteolytic and/or lipolytic.

The color indicator of the present invention will be sensitive to small amounts of acid production in milk to allow the early detection of potentially degrading organisms. The indicator should display a perceptible color change over the pH range between fresh milk and milk which is about to undergo protein precipitation. Moreover, the color indicator should function with all types of acid-producing degradation, in particular those caused by lactic bacteria and sweet-curd forming bacteria. While the particular color response to each of these spoilage mechanisms may

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differ, the color response should be repeatable for each of the mechanisms. In this way, an observed color transition allows the person testing the sample to visually assess suitability of the sample for testing. Samples having unacceptable bacterial levels which result in degradation of the sample can thus be identi¬ fied and separated prior to testing.

Suitable color indicators will display color transition over the pH range from about 6.7 (the pH of fresh milk) to about 4.6 (the isoelectric point of casein). Color transition should be most apparent in the pH range from about 6.1 to 5.1 where protein precipitation is most likely to commence. Protein precipitation as a result of lactic acid formation by lactic bacteria usually begins at a pH in the range from about 5.1 to 5.2, while precipitation resulting from sweet-curd bacteria is more variable and can occur at a- pH as high as about 5.9 to 6.7. ^■

Conveniently, the color indicator will provide a strong coloration of the fresh milk product as an indication that the preservative composition has been added. In this way, accidental consumption of the preserved milk samples will be avoided.

Suitable color indicating compounds include bromcresol purple, chlorophenol red, nitrazine yellow and bromothymol blue which are available from J.T. Baker Chemical Co., Phillipsburg, N.J., as product nos. C950, F334, R778 and D470, respectively. Combina¬ tions of the color indicators are also suitable and can provide enhanced color transition over a desired segment of the pH range.

The preservative compound of the present invention will be chosen to be compatible with the color indicating compound. In particular, the preserva- tive should not interfere with the Coloration or the color change brought about by the color indicating compound. Suitable preservative compounds, which

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display little or no coloration, include boric acid, sodium omadine, and 2-bromo-2-nitropropane-l,3-diol (BND). The latter compound is available under the tradename Bronopol from Boots Chemical Company, Ltd. , London, United Kingdom.

The preferred embodiment of the present inven¬ tion employs bromcresol purple as the color indicator in combination with 2-bromo-l-nitropropane-l,3-diol (BND) as the preservative compound. The BND is colorless when added to milk. The addition of the bromcresol purple, however, causes a deep blue colora¬ tion of the milk at pH 6.7 when the milk is fresh. Such coloration informs those handling the milk samples that the preservative composition has been added and that the milk is unsuitable for consumption.

The preservative composition employing brom¬ cresol purple provides the following color transition.

Color Transition of Bromcresol Purple in Milk p_H Color Action

6.7-6.3 Blue to blue gray Sample normal; proceed with test

6.3-5.3 Gray-green with Examine sample for traces of yellow graininess, curd to yellow-green and/or whey formation; if none, test sample.

5.3-4.6 Yellow with no Discard sample trace of green

Using the preferred bromcresol purple formula¬ tion of the present invention, personnel testing the milk samples can decide on the appropriate action depending on the color of the sample, as set forth above. When the sample is blue or blue-gray, the pH is

such that no substantial degradation of the sample is likely to have occurred. When the color displays traces of yellow in combination with green, the pH is in the range where sweet-curd degradation or acid proteolysis may have occurred, depending on the micro¬ organism involved. It is necessary to examine the sample for signs of protein precipitation, i.e., graini- ness, curd formation, and whey separation. If any of these are apparent, the sample should be discarded. If not, the sample may be tested. Finally, if the sample is yellow with no traces of green, the pH is such that lactic acid precipitation will almost certainly have occurred and the sample should be discarded.

The indicator compound and preservative compound of the present invention will normally be combined with an inert solid or liquid carrier. Conveniently, they will be combined.with a solid carrier and formed into tablets of an appropriate size for treating the contemplated milk samples. The relative and absolute portions of the preservative, color indicator and vehicle will depend on the size of the milk sample to be treated as well as on the effective concentrations of the active ingredients and the desired tablet size. Bromcresol purple should be added to the tablet in an amount sufficient to provide a concentration of at least O.Olmg/ml when added to the milk sample, usually in the range from 0.015 to 0.05mg/ml. The effective ranges for the various preservative compositions are well known in the art. The inert carrier will typically be a soluble salt, such as sodium chloride or potassium chloride, selected based on availability and low cost. Additional components may be added to the tablet, including anti- caking agents and lubricants which facilitate tablet formation in a machine and dispersing agents which enhance the solution of the tablets in milk. Exemplary anticaking agents, lubricants and dispersing agents are set forth below.

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An exemplary tablet formation intended for treatment of 50ml milk samples is as follows:

Exemplary Tablet Formulation

Effective 6 Preferred6 Component Range Formula

Bromcresol purple 0.50-1.0% 0.75%

BND 5.00-10 % 10.00%

Vehicle ² 0.00-95 % 47.25% Anticaking agent 0.25-2.0% 1.00% Lubricant 4 0.25-2.0% 1.00%

Dispersing agent 0.25-50 % 40.00%

1. Based on treatment of 50ml milk sample. 2. Usually KC1 and/or NaCl.

3. Silica, silicon dioxide, and combinations thereof.

4. Stearic acid, lauric acid, oleic acid, boric acid, and combinations thereof.

5. Glucono-δ-lactone. 6. Percentage based on 120mg tablet.

The exemplary tablet can be produced as follows. The crystalline components, e.g., BND, KC1, NaCl, and glucono-δ-lactone, are combined with one-half the boric acid and passed through a 20 mesh (Tyler standard) screen. The powder components, e.g. silicon dioxide, and stearic acid, are combined with the remaining boric acid passed through a 40 mesh (Tyler standard) screen. The bromcresol purple is ground, typically using a mortar and pestal, with a small quantity of the crystalline components blend added. The remaining crystalline components, the bromcresol purple and the powder components are then combined and mixed in a tumbler mixer until the composition is uniform, usually taking from 20 to 40 minutes. The

composition may then be compressed into tablets having a desired size.

The following experiments are offered by way of example and not by way of limitation.

EXPERIMENTAL

Samples (50ml) of fresh raw milk and fresh homogenized milk having bromcresol purple added (18mg/l) were inoculated with a fresh milk culture of Streptococcus cremoris (4% by volume). The samples were allowed to incubate at 88°F for various periods of time, after which they were quickly immersed in an ice water bath. Consistency, color, pH, percent titratable acidity (%TA), and cell density (colony forming units/ml) were determined for each sample. The results are set forth in Table 1.

Table 1

Incubation Period (hours)

Sample Measurement 0 1.5 2.0 2.5 3.0 3.5 4.0

Raw pH 6.45 6.30 6.10 5.9? 5.79 5.65 5.08 milk %TA 0.19 0.23 0.26 0.30 0.34 0.38 0.48 cfu/ml* 0.66 1.50 2.10 3.50 5.70 8.20 5.60 color blue gray- yellow- yellow- light light yellow green green green yellow yellow green green consistency liquid liquid liquid liquid liquid liquid ppt.

Homo. pH 6.45 6.34 6.12 5.98 5.77 5.27 4.85 milk %TA 0.19 0.22 0.26 0.30 0.35 0.46 0.54 cfu/ml* 0.76 1.30 2.40 3.30 6.30 7.80 7.50 color blue gray- yellow- yellow- light yellow bright green green green yellow- yellow green consistency liquid liquid liquid liquid liquid ppt. ppt.

* (xlO ⁸ )

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Samples (50ml) of fresh raw milk and fresh homogenized milk having bromcresol purple added (18mg/l) were treated with lactic acid to simulate the acid development caused by bacterial growth. Consistency, color, pH and %TA were determined for each sample. The results are set forth in Table 2.

Table 2

Lactic Acid (10%,ml)

Sample Measurement 0 0.5 1.0 2.0 3.0 4.0 -5.0

Raw pH 6.56 6.45 6.34 6.09 5.82 5.59 5.27 milk %TA 0.16 0.19 0.22 0.28 0.34 0.40 0.46 color blue blue gray- yellow- yellow- yellow- light green green green green yellow consistency liquid liquid liquid liquid liquid liquid ppt.

Homo. pH 6.68 6.49 6.34 6.06 5-77 5.54 5.11 milk %TA 0.15 0.18 0.21 0.27 0.34 0.40 0.47 color blue blue gray- yellow- yellow- yellow- yellow green green green green consistency liquid liquid liquid liquid liquid liquid ppt.

These results indicate that the color indicator of the present invention provides a useful indication of the incipient bacterial degradation of milk. Such indication allows personnel testing the milk to screen individual samples and to eliminate unsuitable samples prior to testing.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will ^" be obvious that certain changes and modifications may be practiced within the scope of the appended claims.