Pimaricin and Mycostatin

for

R e t a r d i n g C o t t a g e Cheese S p o i l a g e 1 K. Pvl,NtLSON, 2 K. M. SHAHAN! J. R. VAKIL, and A. KILARA Department of Food Science and Technology University of Nebraska, Lincoln, NB 68503 ABSTRACT

procedures during manufacture, packing, and distribution of Cottage cheese, several workers have suggested using cultured dressings (6, 9), citric acid (7), vitamin K s (12), potassium sorbate (2), and antibiotics for improving the shelf-life of Cottage cheese (6). Since the discovery of antibiotics and other antimicrobial agents, food technologists and microbiologists have shown the effectiveness of these compounds in retarding microbial spoilage of perishable foods. At present the Food and Drug Administration does not permit the use of antibiotics for food preservation. However, two antifungal agents, pimaricin and mycostatin, have been described as essentially nontoxic when ingested orally (5). Also, the high order of activity against yeast and mold and loss of biological activity when ingested make them attractive for preserving cultured foods against fungal spoilage (5, 10). Shahani et al. (10) observed that the two antifungal agents added to Cottage cheese through wash water considerably extended the shelf-life of cheese. These antifungal agents were effective in controlling spoilage caused by yeast and mold but had no effect against bacterial spoilage. In a later study, Shahani and Goldberg (11) reported that pimaricin retarded the mycotoxin production of several molds more effectively than the growth of the molds. This study was to determine the effectiveness of pimaricin and mycostatin in retarding spoilage of cheese which was experimentally contaminated with yeast or mold.

Two antifungal agents, pimaricin and mycostatin, added to Cottage cheese through the wash water at concentrations of 20, 50, or 100/~g/ml of wash water or added through the cheese dressing at 1, 2, or 5 /lg/g retarded the growth of Aspergillus niger and Saccbaromyces cerevisiae and improved the shelf-life of the cheese. In general, cheese with highest concentration of antifungal agent and stored at lowest temperature had best keeping quality. Pimaricin was slightly more effective than mycostatin in inhibiting fungi; inhibition was greater if the antifungal agents were added to the cheese dressing and the cheese was stored at low temperature; and A. niger was more sensitive to the inhibitors than S. cere-

visiae. INTRODUCTION

Chief defects that develop in Cottage cheese during storage are caused by the growth of psychrotrophic microorganisms, and spoilage caused by yeast and mold has been among the primary defects of Cottage cheese (3). Foster et al. (4) report that some Cottage cheese samples may contain up to 24 million yeast cells/g and suggest that the porous structure of Cottage cheese provides an ideal environment for fungal growth. Bonner and Harmon (1) isolated Rbodotorula tiara, Torula candida, and Geotrichum candidurn, Mucor plurnbcus, and Penicillium frequentans from spoiled Cottage cheese. Various methods have been developed to improve the keeping quality of cheese during storage. Besides recommending strict sanitary

Received April 22, 1974. ~Published with the approval of the Director as Paper No. 3782, Journal Series, Agricultural Experiment Station, Lincoln, Nebraska. Research work was conducted under Project No. 16-17. 2Present address: Animal Sciences Department, University of Vermont, Burlington.

MATERIALS AND METHODS Manufacture of Cottage Cheese and Incorporation of Antifungals and Fungi

Cottage cheese was manufactured in a 1140 liter vat by the overnight long set procedure with a 1.5% culture inoculation. After the curd was washed once with regular untreated city water at 15.6 C, it was divided into two lots. One lot was a control and was rewashed with regular water. The other lot was washed with

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COTTAGE CHEESE SPOILAGE water containing 20, 50, or 100/ag of pimaricin or mycostatin/ml. When completely drained, both Iots were creamed and packaged in regular 340 g cartons previously dipped in a 200 ppm chlorine solution. Cultures of Aspergillus niger mold or Saccbaromyces cerevisiae yeast grown overnight were added to the experimental samples at .1 ml cultured broth suspended in .9 ml sterile water per 340 g carton. Cheese samples were stored at 4.4, 10.0, or 15.6 C and examined daily until spoilage was detected. Keeping quality or spoilage of the cheese samples was determined by organoleptic and visual examination by qualified judges who were not aware of the identity of the samples. The next phase of the study was to determine the effect of adding pimaricin or mycostatin directly to the cheese through the cheese dressing. Cheese was manufactured, washed with regular water, and contaminated with yeast or mold by the methods described above• Pimaricin or mycostatin was added through the cheese dressing to yield the final concentrations of 1, 2, or 5 /.tg/g of cheese. The cheese was then stored at 4.4, 10.0, or 15.6 C and at periodic intervals judged organoleptically as described above. RESULTS AND DISCUSSION Retardation of Spoilage of Cheese Treated with Antifungal Agents Through Wash Water

Treatment of Cottage cheese with pimaricin

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and mycostatin through the wash water increased its keeping quality (Table 1). At the storage temperature of 4.4 C, cheese treated with 20, 50, or 100/lg pimaricin/ml wash water increased shelf-life 5, 10, or 12.4 days. At higher temperatures of 10 and 15.6 C, cheese samples treated with the antifungal agents also increased shelf-life, but the extent of increase was not as great as at 4.4 C. Essentially, results were similar with mycostatin. In general, however, pimaricin-treated cheese had slightly better keeping quality than mycostatin-treated cheeses. Table 1 also presents data concerning the keeping quality of Cottage cheese washed with antifungal wash water and subsequently contaminated with Aspergillus niger mold. The control Cottage cheese samples experimentally contaminated with the mold spoiled within 11.6, 8.0, and 4.0, days at 4.4, 10.0 and 15.6 C storage. The higher the temperature of storage the shorter the keeping quality, evidently because of the relatively rapid multiplication of the contaminant. However, when the cheese had been washed with antifungal wash water (prior to the addition of mold), it had a comparatively better keeping quality. Washing of the cheese with pimaricin (20, 50, and 100 /ag/ml of wash water) increased shelf-life of the samples by 6.7 to 12.7 days at 4 C, 2.0 to 6.0 days at 10 C, and 2.0 to 4.3 days at 15.6 C. Cheese samples washed with mycostatin wash water followed a similar trend.

TABLE 1. Effect of primaricin and mycostatin added through wash water upon the keeping quality of Cottage cheese.

Samplea Control Antifungal Control + M Anti + M Control + Y Anti + Y

Antifungal concentration ~g/ml .

.

.

20 50 100 •

.

20 50 100 .

.

.

20 50 100

Days stored before spoilage 4.4 C 16.6 21.6 26.6 29.0 1 1 . 6

18.3 19.3 24.3 13.3 17.6 20.6 23.6

Pimaricin 10.0 C 15.6 C 11.3 13.3 14.6 19.0 8.0 10.0 11.6 14.0 8.0 9.6 11,3 14.3

6.0 10.0 9.3 12.3 4.0 6.0 6.6 8.3 4.3 7.0 7.0 8.0

4.4 C 16.6 18.0 19.6 27.0 11.6 14.3 15.0 20.3 13.3 14.3 17.0 22.6

Mycostatin 10.0 C 15.6 C 11.3 12.0 15.0 17.3 8.0 10.0 11.0 14.6 8.0 9.3 12.0 13.0

6.0 8.0 10,0 11.6 4.0 5.6 6.6 9.0 4.3 5.3 7.3 9.0

aM = Mold (Aspergillus niger), Y : Yeast (Saccbaromyces cerevisiae). Journal of Dairy Science Vol. 58, No. 5

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NILSON ET AL.

As with mold contaminated cheese, control cheese contaminated with Saccbaromyces cerevisiae yeast also spoiled rapidly, within 4.3, 8.0, and 13.3 days at 15.6, 10, and 4.4 C storage (Table 1). Also, when the same cheese had been washed with antifungal wash water prior to contamination with yeast, the samples had a better keeping quality• The pimaricin wash treatment improved the shelf-life by 4.3 to 10.3 days at 4.4 C storage, and at the higher temperatures of storage the increase in shelf-life was not as great. Mycostatin wash water treatment showed similar results. Effect of Antifungal Agents Added Through Cheese Dressing

During the above phase of work, antifungal treatments of Cottage cheese through the wash water left minute amounts of antifungal agents in the cheese. The residual antifungal concentrations in Cottage cheese were determined by the method of Pagano and Stander (8) with slight modifications. This indicated that only a small portion of the antifungal agent was retained in the cheese and the rest was drained out with the wash water. Therefore, we determined the effect of adding definite amounts of pimaricin or mycostatin directly to the cheese through cheese dressing. When added through the dressing, both antifungal agents, pimaricin and mycostatin, improved markedly the keeping quality of the

cheese (Table 2). At the storage temperature of 4.4 C, pimaricin added at 1, 2, or 5 #g/g improved the shelf-life of the cheese by 2.3, 18.6, and 26.7 days. At the higher storage temperatures of 10.0 and 15.6 C, the improvement in the keeping quality was not as great. Likewise, mycostatin improved considerably the keeping quality of Cottage cheese and showed the highest beneficial effect at the lowest storage temperature of 4.4 C. Both of the antifungals retarded the growth of mold and yeast in the experimentally contaminated Cottage cheese and thereby improved the keeping quality of the cheese (Table 2). The relationship between concentration of the antifungal added and increase in shelf-life of the Cottage cheese was direct. Further, the maximum beneficial effect of the antifungal agents was at 4.4 C, next at 10.0 C, and the least at 15.6 C. As in the earlier work both antifungals appeared to be more effective against A. niger than against S. cerevisiae. Under comparable conditions pimaricin appeared to be slightly more effective in retarding spoilage than mycostatin. ACKNOWLEDGMENT

Appreciation is expressed to Professors T. A. Evans, L. K. Crowe, and P. A. Downs for assistance in judging the cheese samples. Skillful technical assistance of Miss Clara Zoz is thankfully acknowledged. Pimaricin and mycostatin

TABLE 2. Effect of pimaricin and mycostatin added through cheese dressing upon the keeping quality of Cottage cheese. Days stored before spoilage Samplea Control Antifungal Control + M Anti + M Control + Y Anti + Y

Antifungal concentration tzg/g .

.

.

1 2 5 .

.

.

1 2 5 •

.

.

1 2 5

4.4 C 16.6 18.3 35.2 43.3 11.6 17.3 29.3 32.6 13.3 15.7 21.6 33.7

Pimaricin 10.0 C 15.6 C 11.3 13.0 21.3 23.6 8.0 11.3 17.6 22.6 8.0 10.0 11.7 17.7

6.0 6.0 11.0 8.6 4.0 6.0 5.4 11.0 4.3 4.6 6.7 7.0

aM = Mold (Aspergillus niger), Y = Yeast (Saccbaromyces cerevisiae). Journal of Dairy Science Vol. 58, No. 5

4.4 C 16.6 20.3 25.2 37.6 11.6 19.3 22.3 29.6 13.3 22.3 24.0 27.0

Mycostatin 10.0 C 15.6 C 11.3 13.6 16.0 16.3 8.0 11.3 13.6 16.0 8.0 11.7 13.3 16.3

6.0 6.0 7.7 9.3 4.0 5.7 6.0 8.6 4.3 6.0 5.6 7.0

COTTAGE CHEESE SPOILAGE were supplied b y G i s t - B r o c a d e s C o m p a n y (Royal Dutch Fermentation Industries) and S q u i b b I n s t i t u t e for Medical Research.

REFERENCES 1. Bonner, M. D., and L. G. Harmon. 1957. Characteristics of organisms contributing to spoilage in Cottage cheese. J. Dairy Sci. 40:1599. 2. Collins, E. B., and H. H. Moustafa. 1969. Sensory and shelf-life evaluations of Cottage cheese treated with potassium sorbate. J. Dairy Sci. 52:439. 3. Davis, P. A., and F. J. Babel. 1954. Slime formation on Cottage cheese. J. Dairy Sci. 37:176. 4. Foster, E. M., F. E. Nelson, M. L. Speck, R. N. Doetsch, and J. C. Olson, Jr. 1957, Page 355 in Dairy Microbiology. Prentice Hall Inc. Englewood Cliffs, NJ. 5 Laboratory Applications of Mycostatin. 1955. Medical Div., The Squibb Inst. Med. Res., New Brunswick, NJ. 6. Mather, D. W., and F. J. Babel. 1959. Inhibition of certain types of bacterial spoilage in creamed

7. 8.

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Cottage cheese by the use of a creaming mixture prepared with Streptococcus citrovortis. J. Dairy Sci. 42:1917. Olson, H. C., and O. D. Ball. 1957. Control of bacterial spoilage in Cottage cheese. J. Dairy Sci. 40:1389. (Abstr.) Pagano, J. F., and H. Stander. 1955. Bioassay of nystatin (mycostatin) in body fluids in therapy of fungus diseases. Page 186 in An international symposium. T. H. Sternberg and V. D. Newcommer, eds. Little Brown Co., Boston, MA. Rhodes, M. D. 1953. A study of lipolytic and proteolytic spoilage of Cottage cheese. M. S. Thesis, Texas Tech. Coll., Lubbock, TX. Shahani, K. M., K. M. Nilson, and P. A. Downs. 1959. Effect of antifungal antibiotics upon the keeping quality of Cottage cheese. Proc. XV Int. Dairy Cong. 2:926. Shahani, K. M., H. S. Goldberg. 1972. Inhibitory effect of pimaricin upon mold growth, metabolism, and toxin production. Page 22 in Proc. IUPAC Syrup. Control of mycotoxins, Aug. 21-22, Goteborg, Sweden. Yang, H. V., W. F. Steele, R. W. Stein, R. F. Cain, and R. O. Sinnhuber. 1958. Vitamin K 5 as a food preservative. Food Technol. 12:501.

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Pimaricin and mycostatin for retarding cottage cheese spoilage.

Two antifungal agents, pimaricin and mycostatin, added to Cottage cheese through the wash water at concentrations of 20, 50, or 100 mug/ml of wash wat...
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