and improve the stability of the standards, add 1.0 ml. of 0.05 percent copper sulfate solution 5-a to each.

Add 5.0 ml, of color dilution buffer 1-c and add water to bring the volume to 10.0 ml. Add 4 drops (0.08 ml.) of BQC 4, mix, and allow to develop for 30 minutes at room temperature. If the butyl alcohol extraction method is to be used in the test, extract

the standards as described under III Conducting the Test.

Read the color intensities with a photometer, subtract the value of the blank from the value of each phenol standard, and prepare a standard curve (straight line). When the standards are to be used for visual comparisons they should be stored in a refrigerator.

II. Sampling-1. Hard cheese. Take а sample from the interior with a clean Roquefort trier, place in a small tube, stopper the tube, and keep it in a refrigerator.

2. Soft and semisoft ripened cheese. Harden the cheese by chilling it in the freezing chamber of a refrigerator. Taking special precaution to avoid contaminating the sample with phosphatase that may be present on the surface, use either of the following methods for sampling:

a. Cut a portion from the end of the loaf or from the side of the cheese, extending in at least 2 inches if possible or to a point somewhat beyond the center in the case of a small cheese. Cut a slit 1⁄4 to 1⁄2 inch deep at least halfway around the portion and midway between the top and bottom. Break the portion into two parts, pulling it apart careful not to contaminate the freshly exposed, broken surface. Remove the sample from the freshly exposed surface at or near the center of the cheese.

b. Remove the surface of the area to be sampled-e. g., the end and the adjacent sides with a clean knife or spatula, to a depth of 4 inch. Clean the instrument and hands with hot water and phenol-free soap so that it breaks on a line with the slit, being and wipe them dry. Remove the freshly exposed surface to a similar or greater depth and repeat the cleaning. Then take the sample from the center of the freshly exposed area, preferably at or near the center of the cheese in the case of a small cheese.

3. Process cheese, spreads, etc. Take the sample from beneath the surface with a clean knife or spatula.

Avoid the use of samples contaminated with mold.

4. Preservation. If a preservative is necessary, put 1 to 3 ml. of chloroform in the container, cover with a plug of cotton, insert sample and stopper container tightly. Label preserved samples, "Poison-Preservative added."

III. Conducting the test. 1. Weigh, on a clean balance pan or watch glass, a 0.50-gm.

sample (preferably two samples in duplicate) and place in a culture tube 16 or 18 x 150 mm. Similarly, weigh another sample and place in a tube as a control or blank. If the cheese is sticky, weigh the sample on a piece of wax paper about 1 x 1 inch and insert the paper with the sample into the tube. Macerate the blank and the test with a glass rod about 8 x 180 mm.

2. Add to the blank 1.0 ml. of the appropriate (Table 1) barium buffer 1-a (without substrate added), macerate with the rod, leave the rod in the tube, heat for about a minute to at least 85° C. (185° F.) in a beaker of boiling water with the beaker covered so that the entire tube becomes heated to approximately 85° C., cool to room temperature, and macerate again with the rod.

3. Add the test 1.0 ml. of the appropriate (Table 1) barium buffer substrate 2-a or 2-b. and macerate.

From this point, treat the blank and the test in a similar manner.

Add 9.0 ml. of the appropriate barium buffer substrate 2-a or 2-b (total, 10.0 ml. added), and mix. The rod may be left in the tube during incubation; or, if removing it at this point, cut a piece of filter paper approximately 1 x 1 inch, wrap and hold it tightly around the rod, rotate the rod while withdrawing it from within the tube so as to wipe the rod clean, insert the paper with the adhering fat into the tube, and stopper the tube.

4. Incubate in a water bath at 37°-38° C. (99°-100° F.) for 1 hour, mixing or shaking the contents occasionally.

5. Place in a beaker of boiling water for nearly a minute, heating to 85° C. (185° F.), and cool to room temperature.

6. Pipet in 1.0 ml. of the zinc precipitant 3-b for ripened cheese or the zinc-copper precipitant 3-a for unripened cheese, and mix thoroughly (pH of mixture, 9.0-9.1).

7. Filter (5-cm. funnel, 9-cm. Whatman No. 42 or No. 2 paper recommended), and collect 5.0 ml. of filtrate in a tube, preferably graduated at 5.0 and 10.0 ml.

8. Add 5.0 ml. of color-development buffer 1-b (pH of mixture, 9.3-9.4).

1

9. Add four drops 1 of BQC 4, mix, and allow the color to develop for 30 minutes at room temperature.

10. Determine the amount of blue color by either of two methods:

a. With a photometer. Read the color intensity of the blank and that of the test, subtract the reading of the blank from that of the test, and convert the result into phenol equivalents by reference to the standard curve described under "Phenol standards." The butyl alcohol extraction method is ordinarily unnecessary when using a photometer.

b. With visual standards. For quantitative results in borderline instances, e. g., tests yielding 0.5 to 5 units of color, extract with butyl alcohol 5-b. Add 5.0 ml. of the alcohol and invert the tube slowly several times Centrifuge if necessary to increase the clearness of the alcohol layer. Compare the blue color with the colors of standards in the alcohol.

With samples yielding more than 5 units, compare the colors in aqueous tests with those of aqueous standards.

11. Dilution method for quantitative results. In tests that are observed during color development to be strongly positive, e. g., 20 units or more, in which four drops of BQC may be much less than sufficient to combine with all of the phenol, pipet an appropriate proportion of the contents into another tube, make up to 10.0 ml. with colordilution buffer 1-c, and add two drops more of BQC in the case of unripened cheese or four drops in the case of ripened cheese. With each test, dilute and treat the blank in the corresponding manner. Dilute each strongly positive test thus until the final color is within the range of the standards or photometer. Allow 30 minutes for color development after the last addition of BQC, and make the reading at the end of the 80minute period. Multiply, for example, by 2 for a 5+5 dilution, 10 for a 1+9 dilution, and 50 for a 1+9 followed by a 2+8 dilution.

Alternatively, to reduce the amount of yellow off color, add two instead of four drops of BQC after each dilution, and allow the color to develop. Then test the completeness of color development by adding a third drop; repeat the dilution procedure until the addition of an extra drop does not cause any further increase in the amount of blue color.

12. Calculation and evaluation of results. When using 0.5 gm. of sample and adding a total of 11.0 ml. of liquid, multiply the value of the reading by 1.1 to convert it to units of color or phenol equivalents per 0.25 gm.

1 For merely detecting underpasteurization. in testing unripened cheese, two drops is sufficient, provided the visual standard are prepared likewise with two drops.

of cheese. The result may, if desired, be converted to phenol equivalents per 1 gm. by multiplying by 4.4.

To read

IV. Photometric determination. the color in aqueous solution, use a filter with maximum light transmission in the region of 610 mu wave length.

To read the color in butyl alcohol, extract the color as described above. If necessary, centrifuge the sample for 5 minutes to break the emulsion and to remove the moisture suspended in the alcohol layer. A Babcock centrifuge can be adapted for this purpose by making special tube holders as follows: Slice a section 4 inch thick from a rubber stopper of suitable diameter to fit in the bottom of the centrifuge cup. Glue together two cork stoppers of appropriate diameter. bore through the center a hole of proper size to hold the tube snugly, and insert the double-cork section into the cup. After centrifuging, remove nearly all of the butyl alcohol by means of a pipet with a rubber bulb on the top end. Filter the alcohol into the photometer cell and read with a filter with maximum light transmission in the region of 650 mμ wave length.

If more than approximately 4 ml. of butyl alcohol is required for the photometer used. conduct the test in a larger tube and extract the color, in both the test and the standards. with the necessary quantity of butyl alcohol rather than with 5 ml. specified above.

V. Precautions. The length of time that the crystalline disodium phenyl phosphate and the BQC powder will remain stable can he increased greatly by keeping them in the freezing chamber of a refrigerator, and by keeping them dry.

The glassware, stoppers, and sampling tools should be scrupulously clean, and it is desirable to soak them in hot, running water after cleaning.

The solid barium hydroxide and the barium buffer must be kept stoppered tightly to prevent absorption of carbon dioxide. Phenolic contamination from plastic closures on reagent bottles has been encountered and therefore the use of plastic closures should be avoided. Rubber stoppers should not be used in flasks in which butyl alcohol is stored. Glass or cork stoppers should be used.

VI. Modifications for different cheeses Different kinds of cheese and cheeses of different ages have different buffering capacities, and therefore some of them require modification of concentrations of the reagents. The modifications of the barium buffer needed to produce optimal pH conditions during incubation (9.85-10.20), and of the precipitant to yield uniformly clear filtrates and to minimize interference during color development under optimal pH conditions (9.3-9.4), are specified in Table I.

With some samples, especially those of unknown history, slight deviations from the optimal pH range may occur, but such deviations do not very materially affect the re

sults. For example, pH values as low as 9.6 or as high as 10.35 during incubation have been found to result in an average decrease of not more than 20 percent below the maximum in the quantity of phenol liberated. The use of the 25-11 buffer substrate with samples for which the 27-11 buffer substrate is specified yields pH values not lower than 9.8.

In testing cheese of unknown history or age, information as to the percentage of solids, especially the nonfat solids, is useful as an indication of the correct buffer to use; cheese with a relatively high percentage of nonfat solids generally requires the use of a relatively concentrated buffer to adjust the pH of the mixture correctly.

For precise quantitative results on unknown samples, adjust the pH to 10.0-10.05 for the incubation.

Cottage cheese curd is heated in the presence of considerable acid during manufac ture, and therefore its phosphatase values are comparatively low. Alternatively, to increase the sensitivity of the test on cottage cheese, apply the following modifications: Use a 1.0-gm. sample, 27-11 buffer substrate 2-hour incubation, and 6.0-0.1 precipitant.

(g) (1) If cheddar cheese in sliced or cut form contains an optional mold-inhibiting ingredient as specified in paragraph (d) of this section, the label shall bear the statement".

retard mold growth” or “.

added to added

as a preservative," the blank being filled in with the common name or names of the mold-inhibiting ingredient or ingredients used; e.g., "Sorbic acid and potassium sorbate added to retard mold growth."

(2) Wherever the name of the food appears on the label so conspicuously as to be easily seen under customary conditions of purchase, the statement specified in this section, showing the optional ingredient used, shall immediately and conspicuously precede or follow such name, without intervening written. printed, or graphic matter.

[24 F.R. 6478, Aug. 12, 1959, as amended at 27 F.R. 3005, Mar. 30, 1962; 28 FR. 5420, June 1, 1963; 29 F.R. 15811, Nov. 25, 1964; 30 F.R. 1253, Feb. 5, 1965; 32 F.R. 410, Jan. 14, 1967] § 19.502 Cheddar cheese for manufacturing; identity.

Cheddar cheese for manufacturing conforms to the definition and standard of identity prescribed for cheddar cheese by 19.500, except that the milk is not pasteurized, curing is not required, and the provisions of paragraph (d) of that section do not apply.

[24 F.R. 6481, Aug. 12, 1959]

§ 19.503

Low sodium cheddar cheese; identity; label statement of optional ingredients.

Low sodium cheddar cheese is the food prepared from the same ingredients and in the same manner prescribed in § 19.500 for cheddar cheese and complies with all the provisions of § 19.500, including the requirements for label statement of optional ingredients, except that:

(a) Salt is not used. Any safe and suitable ingredient or combination of ingredients that contains no sodium and that is recognized as a salt substitute may be used.

(b) Sodium sorbate is not used.

(c) It contains not more than 96 milligrams of sodium per pound of finished food.

(d) The name of the food is "low sodium cheddar cheese." The letters in the words "low sodium" shall be of the same size and style of type as the letters in the words "cheddar cheese,” wherever such words appear on the label.

(e) If a salt substitute as provided for in paragraph (a) of this section is used, the label shall bear the statement added as a salt substitute," the blank being filled in with the common name or names of the ingredient or ingredients used as a salt substitute.

(f) Low sodium cheddar cheese is subject to the regulations for foods for special dietary uses promulgated under the provisions of section 403 (j) of the Federal Food, Drug, and Cosmetic Act. [35 F.R. 530, Jan. 15, 1970]

§ 19.505 Washed curd cheese, soaked curd cheese; identity; label statement of optional ingredients.

(a) Washed curd cheese, soaked curd cheese, is the food prepared from milk and other ingredients specified in this section, by the procedure set forth in paragraph (b) of this section, or by another procedure which produces a finished cheese having the same physical and chemical properties as the cheese produced when the procedure set forth in paragraph (b) of this section is used. It contains not more than 42 percent of moisture, and its solids contain not less than 50 percent of milk fat, as determined by the methods prescribed in § 19.500 (c). If the milk used is not pasteurized, the cheese so made is cured at a temperature of not less than 35° F. for not less than 60 days.

(b) Milk, which may be pasteurized or clarified or both, and which may be warmed, is subjected to the action of harmless lactic-acid-producing bacteria, present in such milk or added thereto. Harmless artificial coloring may be added. Sufficient rennet, or other safe and suitable milk-clotting enzyme that produces equivalent curd formation, or both, with or without purified calcium chloride in a quantity not more than 0.02 percent (calculated as anhydrous calcium chloride) of the weight of the milk, is added to set the milk to a semisolid mass. The mass is so cut, stirred, and heated with continued stirring, as to promote and regulate the separation of whey and curd. The whey is drained off, and the curd is matted into a cohesive mass. The mass is cut into slabs, which are so piled and handled as to promote the drainage of whey and the development of acidity. The slabs are then cut into pieces, cooled in water, and soaked therein until the whey is partly extracted and water is absorbed. The curd is drained, salted, stirred, and pressed into forms. A harmless preparation of enzymes of animal or plant origin capable of aiding in the curing or development of flavor of washed curd cheese may be added during the procedure, in such quantity that the weight of the solids of such preparation is not more than 0.1 percent of the weight of the milk used.

(c) For the purposes of this section: (1) The word "milk” means cow's milk, which may be adjusted by separating part of the fat therefrom or by adding thereto one or more of the following: Cream, skim milk, concentrated skim milk, nonfat dry milk, water in a quantity sufficient to reconstitute any concentrated skim milk or nonfat dry milk used.

(2) Milk shall be deemed to have been pasteurized if it has been held at a temperature of not less than 143' F. for a period of not less than 30 minutes or for a time and at a temperature equivalent thereto in phosphatase destruction.

Washed curd cheese shall be deemed not to have been made from pasteurized milk if 0.25 gm. shows a phenol equivalent of more than 3 micrograms when tested by the method prescribed in § 19.500 (f).

(3) During the cheese-making process the milk may be treated as provided in § 19.500 (e) (3).

(d) Washed curd cheese in the form of slices or cuts in consumer-sized pack

ages may contain an optional mold-inhibiting ingredient consisting of sorbic acid, potassium sorbate, sodium sorbate, or any combination of two or more of these, in an amount not to exceed 0.3 percent by weight, calculated as sorbic acid.

(e) (1) If washed curd cheese in sliced or cut form contains an optional moldinhibiting ingredient as specified in paragraph (d) of this section, the label shall bear the statement ". added to retard mold growth" or as a preservative," the blank being filled in with the common name or names of the mold-inhibiting ingredient or ingredients used.

66

added

(2) Wherever the name of the food appears on the label so conspicuously as to be easily seen under customary conditions of purchase, the statement specified in this section, showing the optional ingredient used, shall immediately and conspicuously precede or follow such name, without intervening written, printed, or graphic matter.

[24 F.R. 6481, Aug. 12, 1959, as amended at 25 F.R. 1016, Feb. 5, 1960; 27 F.R. 3005, Mar. 30, 1962; 28 F.R. 5420, June 1, 1963; 30 FR. 1253, Feb. 5, 1965; 32 FR. 410, Jan. 14, 1967] § 19.507 Washed curd cheese for manufacturing; identity.

Washed curd cheese for manufacturing conforms to the definition and standard of identity prescribed for washed curd cheese by § 19.505, except that the milk is not pasteurized, curing is not required, and the provisions of paragraph (d) of that section do not apply. [24 F.R. 6481, Aug. 12, 1959]

§ 19.510 Colby cheese; identity; label statement of optional ingredients.

(a) Colby cheese is the food prepared from milk and other ingredients specified in this section, by the procedure set forth in paragraph (b) of this section, or by another procedure which produces a finished cheese having the same physical and chemical properties as the cheese produced when the procedure set forth in paragraph (b) of this section is used. It contains not more than 40 percent of moisture, and its solids contain not less than 50 percent of milk fat, as determined by the methods prescribed in § 19.500 (c). If the milk used is not pasteurized, the cheese so made is cured at a temperature of not less than 35° F. for not less than 60 days.