Inactivation of Nisin by Pancreatin

TECHNICAL

NOTES

Inactivation of Nisin by Pancreatin Nisin is a polypeptide, produced by certain strains of Streptococcus lactis~ having the unique function of preventing, in minute quantities, the outgrowth of heat-damaged spores (9). One of the first applications of this property to the canning industry was reported by Gillespy (7). Subsequent reports by several investigators have demonstrated the usefulness of nisin as a heatsterilization aid. Wide usage of nisin in canned foods, however, would depend in part upon its toxicological properties. Although the studies of :Frazer et al. (5) demonstrated its safety for use in foods at the levels of proposed treatment, it was thought that a specific study of the effect of pancreatin on the activity of nisin would be of interest. Should it be shown that nisin was inactivated by pancreatin, while medical antibiotics remained active under the same conditions, it could be reasonably assumed that nisin could not influence the intestinal flora and, therefore, would be of no physiological significance in this respect. Whitehead (17), in an early report on the occurrence of a substance causing slowness in cheese starters, reported that this substance was stable toward pepsin but hydrolyzable by trypsin and alkali. Thorpe (16) used 5 mg Bacto trypsin per 1.5 ml phosphate buffer ( p H 6.0), holding at 18 to 20 C for 1 hr to inactivate 10 RU of nisin. This is a level of 333 mg of trypsin per ]00 ml o2 reaction mixture. Campbell and Sniff (3) used Difco Trypsin (1:250) at a p H of 8.0 and a concentration of 5.6 mg per 100 ml of reaction mixture containing 56 RU of nisin per milliliter. MacWalter (10) used Difco Trypsin (1:250) at a p H of 6.0 in phosphate buffer. The National Formulary (11) prescribes the use o£ 4 mg of panereatin per 100 ml of reaction mixture for assaying' purposes and Sahyun (14) recommended 50 mg per 100 ml of reaction mixture at p H 8.0 and 37 C to demonstrate the enzymatic digestion of casein. Methods

Pancreatin, N. ]J~. was chosen as the enzyme source because it contained more of the enzymes likely to occur in pancreatic juice than would be found in Difco Trypsin. The concentration chosen was 25.6 mg pancreatin per 100 ml of reaction mixture. It was determined that levels of 2.5 mg per 100 ml of reaction mixture did not inactivate nisin in 30 min at p H 8.0 at 37 C (Table 1 ) ; however, 5.1 and 12.8 mg were effective. Since the highest level tested would be likely to have the greatest effect on the medical antibiotics to be tested, that level was used in subsequent testing. Nisin activity was determined by the Resazurin reduction method of Friedmann and Ep-

TABLE 1 Effect, of concentration of pancreatin on ~nactivation of nisin at 80 RU per milliliter, pit 8.0, carbonate buffer, and at 37 C Concentration of panereatin per 100 m] reaction mixture (in rag)

RU of nisin activity after digestion for 30 rain

2.5 5.1 12.8 25.6

40 0 0 0

stein (6) with slight modifications, using Strep-

tococcus cremoris as the test organism. The activity of the medical antibiotics was tested by the F D A rapid disc assay for antibiotics in milk (15), using Bacillus subtilis as the test organism. This assay can be completed in about 3 hr. Three buffer systems were tested--phosphate, citrate, and carbonate at 0.01 molar concentrations. It will be noted from Table 2 that the citrate TABLE 2 Influe~we of buffer system on hmetiw~tion of nisi~ at pH 8.0 and 37C RU n i si n activity remaining after digestion

Buffer used (0.01 molar) Carbonate Phosphate Citrate

0.0 hr 34 53 80

0.5 hr 0.0 0.0 27.0

1.0 hr 0.0 0.0 0.0

buffer was slightly inhibitory; therefore, only carbonate and phosphate systems were used in subsequent work on nisin. Phosphate buffer was used with the medical antibiotics. These were digested at o H 8.0, since according to Everett (4) the p H range ot' normal human pancreatic jui(.c is 7.5 to 8.0. Resolts

Table 3 shows the effects of time and p H on the inactivation of nisin in phosphate buffer. At p H 7.0 to 8.5, nisin was completely inactivated in 30 rain. At p H 6.0, nisin was partially inactivated in 1 hr and completely in 2 hr. These results were verified using carbonate buffer. At p H 8.0 and 37 C, the reaction mixture containing 25.6 mg pancreatin per 100 ml 312

TECHNICAL NOTES TABLE 3 Effect of pH and time of digestion of nisin by pancreatin in phosphate buffer

pI-I

(in hours)

Time

RU/ml remaining

6.0

0.0 0.5 1.0 2.0 0.0 0.5 1.0 2.0 0.0 0.5 1.0 2.0 0.0 0.5 1.0 2.0 0.0 0.5 1.0 2.0

64 46 35 0 47 0 0 0 54 0 0 0 53 0 0 0 53 0 0 0

7.0

7.5

8.0

8.5

313

A f t e r incubation of the plates, the diameter of the zones of inhibition was measured and reported in Table 4. I t is a p p a r e n t that all of tile medical antibiotics tested (bacitracin, tetracycline, oxytetracycline, chlortetracycline, penicillin, and tylosin) were not appreciably affected by digestion conditions. Discussion

and 80 R U (2 p p m ) of nisin was found to contain 40 R U at zero reaction time and no detectable activity a f t e r digestion for 15 rain. I t should be pointed out that the sample was removed f r o m the digestion mixture immediately a f t e r combining all ingredients; however, the reaction was not stopped and an additional 15 min was required to p e r f o r m the assay. I t is thus a p p a r e n t that nisin is rapidly inactivated by pancreatin at p H 8.0 and 37 C in earbonate buffer. Certain medieal antibiotics are being used in animal feeds for the purpose of increasing rate of growth. I t is reasonably well established that such stinmlation is due to the influence of the antibiotic on the intestinal flora (13). Several of these antibiotics were p r e p a r e d in phosphate buffer at p H 8.0 and digested with panereatin at a level of 25.6 mg per 100 ml of reaction mixture held at 37 C for 0 to 8 hr. Portions were removed and tested, using the disc assay method. Since this assay requires about 3 hr at 37 C, and since the digestion reaetion was not stopped, zero-digestion time means only that portion removed f o r testing was taken immediately a f t e r combining all ingredients.

Results reported above are not unexpected. Nisin has been reported to be inactivated by trypsin, a component of panereatin. Rate of inactivation is quite r a p i d and, therefore, nisin should be inactivated shortly a f t e r it leaves the stomach (2). Nisin thus would be unable to influence the intestinal flora, and without physiological significance in this respect. Animal feeding studies by H a l l (8) and B a r b e r et al. (1) demonstrated that nisin gave no growth response when fed to pigs. P o r t e r (12), studying several antibiotics, reported that nisin was without effect on growth of chicks. Since nisin is so rapidly degraded, results of these feeding trials would be expected. Medical antibiotics, on the other hand, would be expected to survive pancreatic digestion, since they are known to influence the intestinal flora, and results reported above are those which would be anticipated. Summary

Nis~n is readily inactivated by pancreatin at p i t 8.0 when held at 37 C f o r 15 to 30 rain. I t is reasonable to expect, therefore, that nisin cannot influence the intestinal flora. Bacitracin, tetracycline, oxytetracycline, chlortetracyeline, penicillin, and tylosin were medical antibiotics used to influence the intestinal flora. These were all found to be stable under the same digestion conditions which rapidly inactivated nisin. B. H E I N E M A N N and R. WILLIAMS

Producers Creamery Company Springfield, Missouri References

(1) Barber, R,. S., Brnude, 1~., and Itirseh, A. 1952. Growth of Pigs Given Skim Milk

TABLE 4 Effect of panereatin on inactivation of some medical antibiotics at pH 8.0 in phosphate buffer

Antibiotic Baeitracln Tetracycline Tylosin Penicillin Chlorte'craeycline Oxytetracycline

Level tested 250 nnits/ml 10 #g/mI 10 #g/ml 20 units/ml 10/*g/m] 10 #g/ml

0

Zone of inhibition (in centimeters) after digestion time (in hours) 0.5 3.0 8.0

14 mm ]8 17 26 28 26

11 mm 18 14 26 28 25

11 mm 18 12 26 28 26

11 mm 18 14 27 22 26

314

(2) (3) (4) (5)

(6)

(7)

(8)

(9)

J O U R N A L OF D A I R Y S C I E N C E

Soured with Nisin-Producing Streptococci. Nature, 169: 200. Best, C. It. 1945. The Physiological Basis of Medical Practice. p. 500. Williams & Wi]kins Co., Baltimore, Maryland. Campbell, L. Leon, and Sniff, Ester E. 1959. Effect of Subtilin and Nisin on the Spores of Bacillus coagulans. J. Bacteriol., 77: 766. Everett, Mark R. 1946. Medical Bioehenlistry. 2nd ed., p. 132. Paul B. Hoeber, Inc., New York. Frazer, A. C., Sharrott, M., and Hiekman, J. R. 1962. The Biological Effects of Food Additives. I. Nisin. J. Sei. Food Agr., 13 : 32. Friedmann, Rosa, and Epstein, C. 1951. The Assay of the Antibiotic Nisin by Mea~s of a Reduetase (Resazurin) Test. J. Gem Microbiol., 5: 830. Gillespy, T. G. 1957. Nisin Trials. Research leaflet no. 3. The Fruit and Vegetable Canning and Quick Freezing Research Association, Chipping Campden, Gloucestershire, England. ~Iall, R. It. 1959. Nisin in the Feeding of Bacon Pigs. Aplin and Barrett, Yeovil, Sonlerset~ England. Research report, April 29. Lewis, g. C., Michener, It. D., Stumbo, C. R., and Titus, Dudley S. 1954. Antibiotics

(10)

(11) (12) (13)

(14) (15)

(16) (17)

Accelerating Death of Spores by Moist Heat. Agr. and Food Chem., 2: 298. MacWalter, R. J. 1962. Nisin t~esearch Programme Interim Report, no. 1375. Central Laboratories, United Dairies, Ltd., London, England. National Formulary. 1960. l l t h ed., p. 257. American Pharmaceutical Association, Washington, D.C. Porter, J. W. G. 1954. Antibiotics and Nutrition. J. Appl. Bacterioh, 17: 152. Reyniers, J. A., Luckey, T. D., and Gordon, H . A . 1952. Studies on the Growth Effects of Antibiotics in Germ-Free Animals. Rept. of Colloq., June 4, Lobund Institute, Notre Dame, Ind~ ana. Sahyun, Melville. 1944. Anlino Acids and :Proteins. p. 91. Reinhold Publishing Corp., New York. Standard Methods for the Examination of Dairy Products. 1960. 11th cal., p. 186. American Public Health Association, New York. Thorpe, R. H. 1960. The Action of Nisin on Spoilage Bacteria. J. Apph Bacteriol., 23 : 136. Whitehead, H. R. 1933. A Substance Inhibiting Bacterial Growth Produced by Certain Strains of Lactic Streptococci. Biochem. J., 27: 1792.

Residence Time of Nisin in the Oral Cavity Following Consumption of Chocolate Milk Containing Nisin I t has been demonstrated that nisin is effective as an a d j u v a n t to heat sterilization (3) and, of utmost importance, the toxicology of nisin has been shown to be acceptable for the p r o p o s e d conditions of use (1). Furthermore, consunlption of nisin-containing products would not result in an alteration of the intestinal bacterial flora, because nisin is inactivated by enzymes contained in the intestinal tract (2). However, the question still remained as to whether continued consumption of nisincontaining products might alter the bacterial flora of the oral cavity, making it nisinresistant. F o r such a change to occur, nisin must be present in the oral cavity f o r at least one microbial generation time. This investigation was conducted to determine the length of time nisin may be detected in saliva subsequent to consumption of a product containing nisin. The residence time of penicillin was likewise observed, to determine if residence times of nisin and a medical antibiotic were similar.

Experimental Procedure Nisaplin, m a n u f a c t u r e d by A p l i n & Barrett, Ltd., Yeovil, England, was added to commercially pasteurized chocolate milk such that the product contained a p p r o x i m a t e l y 200 R U / m i l l i liter (about ten times normal levels). Here-

after, the nisin chocolate milk will be r e f e r r e d to simply as chocolate milk. Saliva samples (1-2 ml) were taken from individuals, both before and after drinking chocolate milk, and held at 4 C until the samples were assayed for nisin according to the method of Stumbo et al. (7). Saliva samples were assayed for residual penicillin according to the procedure described for the assay of penicillin in milk (6). Results

To illustrate the residence time of nisin in the oral cavity, chocolate milk was consumed and saliva collected immediately, 30 sec, 1 rain, 5 min, and 10 min a f t e r drinking. D a t a of three trials were averaged and arc presented graphically in F i g u r e 1. W h e n saliva was collected imnlediately a f t e r consumption of the chocolate milk, the concentration of nisin was appreciable---approximately 50 R U p e r milliliter. However, the m a j o r i t y of the nisin disappeared in 1 rain and, a f t e r 5 rain, concentration of the inhibitory substance a p p r o x i m a t e d that of the control saliva. Attention is brought to the fact that the control saliva (saliva collected before consumption of chocolate milk) possessed an inhibitory substance in concentration equal to a p p r o x i m a t e l y 2 R U of nisin p e r milliliter. F o r