Inhibitory Effect of Nisin upon Various Organisms1, 2

INHIBITORY

EFFECT

O F N I S I N U P O N V A R I O U S O R G A N I S M S 1, ..

K. M. SHAHANI Department of Dairy Husbandry, University of Nebraska, Lincoln SUM:MARY

Two nisin-producing strains of Streptococcus lactis were found to vary in their ability to produce the antibiotic. I n the milk medimn containing 5% of nisin-broth, cell multiplication and acid production of two strains of Streptococcus thermophilus were delayed, but upon further incubation their total cell count and acid production were not affected appreciably. Nisin-broth did not inhibit three strains of S. lactis, two strains of Lac~obacillus bulgaricus~ two strains of S. thermophilus, a nfixed lactic culture, and one strain each of Bacillus subtilis and Bacillus cereus. Nisin was not quite as inhibitory as several other antibiotics against the dairy cultures tested. Of 11 strains of Staphylococcus aureus Used, the antibiotic seemed to be active only against the coagulasenegative and weak coagulase-producing organisms. The data presented in this paper indicate that the antibiotic may be considered for use to inhibit certain staphylococci or lactic organisms. Additional work should be conducted to determine the applications of nisin and nisin-producing organisms in the dairy industry.

Nisin, an antibiotic, is a normal metabolite of certain strains of a commonly used cheese starter, Streptococcus lactis. The antibiotic is a large polypeptide with the molecular weight of about 10,000, and it consists of five polypeptide components which vary slightly in their chemical, physical, and biological properties (3, 4). Hawley (9) has reported that nisin is inhibitory against several streptococci, lactobacilli, clostridia, staphylococci, and bacilli; and Mattick and Hirseh (11) observed that the antibiotic has no activity against gram-negative organisms. Several toxicity studies (1, 6, 8) have shown that when the crystalline nisin or milk soured with nisin-producing S. lactis was fed to anfinals, it produced no toxic effects. O'Brien et al. (13) have reported that nisin lowers the thermal resistance of food spoilage organisms and suggested that consideration could be given to using nisin for food preservation. I n the dairy industry, considerable quantities of processed cheese and several other sweet or low-acid cheeses, for example, Swiss, Gouda, Edam, and Parmesan, are spoiled during storReceived for publication February 3, 1960. ~Supported by a grant from the Hatch Fund, Project 589, Department of Dairy Husbandry, University of Nebraska, Lincoln. e Published with the approval of the Director as paper no. 1004:., Jounlal Series, Nebraska Agricultural Experiment Station, Lincoln.

age due to gas-holes or inflation caused by growth of Clostridia or butyric acid fernmntation. McClintock et al. (12) observed that the incorporation of small quantities of nisin in processed Gruyere cheese was found to retard or inhibit such undesirable fermentation. Hawley (9), in his review article, reports that the cheeses in which the antibiotic is produced naturally llave been found to be quite resistant to certain bacterial spoilage defects. Also, stone work has been done relative to the use of nisin-producing streptococci, singly or in combination with other cheese cultures, to prevent the spoilage of cheese (14). As far as is known, comparatively little thought has been given to the determination of the effects of nisin upon the commonly used dairy cultures. This work was initiated, therefore, to investigate the effects of' nisin and nisin-producing streptococci upon the organisms generally found or used in dairy products. EXPERIi~[ElqTALPROCEDURE Several nisin-producing strains of S. lactis, obtained from France, Austria, and England, where much of the previous work on nisin has been done, were used in this work. Preliminary trials revealed that two strains multiplied at a faster rate than the others, and were chosen for further studies. The initial phase of this study was concerned with the comparison of nisin production of the two cultures. The method of Chevalier, Fournaud, and ~¢[ocquot (5), with minor alterations, was used to determine the 827

828

K.M.

SttAHANI

antibiotic production of the cultures. The method involves plating suitable dilutions of a nisin-producing culture and of a nisin-sensitive culture in the same plate, which is then incubated. I t is imperative that the optimum temperature for growth of the sensitive organisms be considerably different from that of the nisinproducing organisms. I n this case, the plate thus prepared is incubated at 22 C, or at the opthnum temperature of the nisin-producing S. lactis, in order for the streptococcus to grow and produce nisin. At this temperature, L. lactis with an optinmm temperature of 32-35 C cannot multiply. After the plate is incubated at 22 C for 24 hr, it is transferred to an incubator at 32 C for 48 hr to permit L. lactis to grow. During the second incubation, the test organism grows normally, except around the colonies of the nisin-producing organisms, and clear zones of inhibition are obtained, as is shown in Figure 1.

FIG. 1. A plate showing the inhibition of Lactobacillus lactis by nlsin produced by Streptococcus lactis. Using this technique, it was observed that when similar dilutions were used for the two nisin-producing organisms ~nd for the test organisms, the two nisin-producing cultures produced nearly the same number of zones or colonies. However, one culture (S. lactis F ) produced bigger zones of inhibition around the colonies than did the other culture (S. lactis T). This was felt to be due to the larger amount of nisin produced by F than by T. To investigate this further, nisin-broths were prepared from ]4- to 16-hr old tomato juice broth suspensions of cultures F and T. Cells were reumved by centrifugation, and the clear broth was heated at 80-90 C for 2 to 3 rain to kill the remaining

live organisms. This heat treatment was considered to have had no effect on the nisin content of the broth, since it has been shown that nisin is highly heat-resistant (7). Ya~Ting concentrations of F and T nisin-broths were introduced in separate broth tubes and were inoculated with 1% broth suspension of L. lactis. Turbidity determinations were made after 14 to 16 hr of incubation at 32 C. It was observed that broth of both the nisin cultures inhibited the growth of L. lactis~ but nearly twice as much broth of T culture was required as F for the same amount of inhibition.

Effect of nisin-broth upon various lactic cultures. Studies were conducted to determine the effect of nisin-broth upon the acid production of three strains of S. lactis, two strains of L. bulgaricus, four strains of S. thermophilus, and a mixed lactic culture. Using the same technique as above, nisin-broth of the nisinproducing culture F was prepared and ndded at the rate of 1- to 18-ml aliquots of sterile milk in test tubes. The tubes were then inoculated with 1 ml of a 12- to 14-hr-old lactic culture prepared in milk. Thus, the tubes contained 5% by volume of the nisin-broth and 5% by volume of inoculum from a culture of the test-organism. The contents of the tubes were mixed thoroughly and incubated at the optinmm temperatures of the test organisms. At periodic intervsls of 6, 16, and 24 hr of incubation, tubes were taken out and titratable acidity wss determined. Four to six trials were conducted with each culture, and average results are presented in Table 1. I t was observed that except for two strains of S. thermophilus, Cultures 8 and 9, the amount of nisin in 1 ml of nisin-broth had no appreciable inhibitory effect upon the three strains of S. lactis, the two strains of L. bulgaricus, the two strains of S. thermophilus, and the mixed culture. The antibiotic inhibited slightly the acid production of two strains of S. thcrmophilus (no. 8 and 9) during the first 6 hr of incubation. However, upon longer incubation, the amounts of acid produced by the cultures in the presence and in the absence of the antibiotic were almost the same. The fact that nisin-broth showed some inhibitory effect upon the acid production of S. thermophilus Y and Z during the initial stages of incuhation and showed no adverse effect upon extended incubation (24 hr) may be explained by a prolongation of the lag phase of the S. thermophilus cultures. This prolongation in the lag phase may be due either to the inhibitory effect of the antibiotic upon the young and/or sensitive bacteria or to the time

INHIBITORY EFFECTS

OF

NISIN

829

TABLE 1 Effect of nisln upon the rate of acid production by vvarious lactic cultures Culture

0 hr

6 hr

16 hr

24 hr

( % Titratable acidity) 1. Str. lactis UN Str. lactis UN ÷ nisin 2. Str. lavtis C Str. lactis C + nisin 3. Str. lactis F~ Str. lactis F~ -b nisin 4. L. bu~garicus C~ L. bulgaricus C~ + nisin 5. L. bulgaric.us b L. bu~garim~ b + nisin 6. Str. thermophilus U Str. thermophilus U + nisin 7. Str. thermophilus X Str. thermaphilus X + nisin 8. Str. thermo~hilus Y Str. thermophilus Y + nisin 9. Str. thermo~hilus Z Str. ther~nophilus Z + nisin

10. Mixed culture F Mixed culture /r + nisin

0.22 0.25 0.23 0'.25 0.25 0.28 0.28 0.30 0.26 0.30 0.21 0.23 0.24~ 0.25 0.23 0.25 0.23 0.25 0.2'1 0.23

required for the culture to get accustomed to the new medium. Also, there exists a possibility that the antibiotic may have lost its activity upon prolonged incubation in the presence of milk solids and of the lactic culture, as found true in terramycin (15). Effect

of

uisin

upon

cell-multiplication.

Hirsch (10) has indicated that nisin is bactericidal rather than baeteriostatie and that it achieves a rapid killing of sensitive bacteria. Also, he suggests that nisin often forms an irreversible complex with the protein moieties of the organisms. Trials were made to determine the effect of nisin upon cell multiplication of the nisin-sensitive L. lactis and S. thermophflus when grown in sterile milk. In this work, the technique used for determining the effect of nisin upon cell multiplication was essentially the same as used in the preceding phase, except that pure nisin ~ was used instead of nisin-broth. Appropriate quantities of aqueous solutions of nisin were added to yield concentrations of 0.5, 1.0, 5.0, and 10.0 units per milliliter. Pure nisin is believed to contain 40 to 50 million Reading units per gram (2, 9). A Reading unit has been described as the amount that will normally just inhibit the growth of the test organism, Streptococcus agalactiae, in 1 ml of broth. Using the standard a C:rystalline nisin was supplied by Alpin and Barret Ltd. Co., England.

0.50 0.48 0.48 0.49 0.45 0.~5 0.79 0.80 0.91 0.94 0.51 0.52 0.46 0.49 0.51 0.44 0.49 0.40 0.46 0.46

0.70 0.6.6 0.73 0.71 0..67 0.6.8 1.42 1.45 1.68 1.60 0.79 0.78 0.69 0.70 0.72 (k69 0.73 0.70' 0.71 0.72

0.81 0.80 0.84 0.85 0..79 0.77 1.88 1.95 1.91 1.89 0.92 0.94 0.87 0.86 0.91 0.90 0.89 0.91 0.90 0..86

plate count method involving duplicate or triplicate plates for each dilution, viable cell counts were determined immediately after adding the antibiotic and then at the end of 6, 12, 18, 24, and 36 hr of incubation. In Figure 2 are presented data representing the average results of five to eight trials conducted with each organism and each concentration level of the antibiotic. Curves I are the control curves, or represent the cell numbers produced by the organisms in the absence of the antibiotic, and Curves I I , I I I , IV, and V represent the cell numbers produced by the organisms in the presence of 0.5, 1.0, 5.0, and 10.0 units of nisin per milliliter, respectively. In the absence of the antibiotic, the organisms grew normally, reaching the maximum counts in 18 to 24 hr and declining thereafter. In the presence of the antibiotic, the viable counts of both the organisms decreased rapidly for a while. There was a direct but improportional relationship between the antibiotic concentration and the reduction in the counts, following which the cultures multiplied normally and paralleled the growth curves of the controls. As little as 0.5 or 1.0 unit of nisin per milliliter effected an immediate slight decrease in the counts, after which the cultures seemed to grow normally. The higher concentrations of tfisin (5.0 and 10.0 units/ml) decreased rapidly the cell counts of the organisms during the first few minutes and continued to do so for the first 6 hr of incubation, but

830

i~. ~M. S H ~ n ~ ]

thereafter the surviving organism nmltiplied normally and proximated the counts of the control samples at the end of 24 to 36 hr. At the end of 36 hr, the total counts of the cultures in the presence of the antibiotic were either just about the same or slightly higher than the control counts. This could be expected, since the cultures in the absence of nisin reached the stationary or declining phase earlier than the cultures in the presence of the antibiotic. Comparison of the inhibitory effect of nisin with other antibiotics. Trials were made to compare nisin with other antibiotics with respect to their inhibitory effect upon various lactic cultures. Individual test tubes containing 19 ml of sterile milk were inoculated with 1 ml of freshly prepared 12- to 16-hr-old lactic cultures. Appropriate amounts of penicillin, streptomycin, aureomycin, or e~Tthromyein were added to obtain concentrations varying between 0.05 and 50.0 ppm of the antibiotics. In nisin, the concentrations varied between 0.5 to 150.0 units per milliliter. The tubes were then incubated for 14 to 16 hr at the respective optimum temperatures of the cultures under study. At Q I,iJ

the end of the incubation period the coagulation of the milk was taken as the criterion of the growth of the cultures. The samples coagulated were assumed to be uninhibited and the samples which showed only partial or no coagulation were considered to be inhibited. Average results of four to six trials with each antibiotic and each culture are presented in Table 2. The table shows the minimum levels of various antibiotics required for inhibiting the organisms. It may be observed that in general the organisms were more sensitive to penicillin, aureomycin, streptomycin, or erythromycin than to nisin. Also, there were observed wide variations in the sensitivities of these organisms to penicillin, aureomycin, streptomycin, erythrolnycin, or nisin. The concentrations of the antibiotics required for the inhibition of the cultures were 0.05 to 0.3 unit of penicillin per m i l l i l i t e r , 0.05 to 0.75 ~g of aureomycin per milliliter, 0.2 to 1.5 tzg of streptomycin per milliliter, or 0.2 to 5.0 /xg of erythronwcin per milliliter. In the case of nisin much higher concentrations of the antibiotic (2.0 to 150 t~

e-, W

0

J er w

0

~-

ao

Z

z

0

O

L. Iocfis

9-

<

<

R

o

S. thermophdus

W 8-

Z

'\, W

Cl0

........ e_ /

~/g

//,,o"

6-

0

5o

_J 4-

0

0

;

2:.

3'6

0

0

6

,2

2,,

36

INCUBATION TIME (HOURS) Fro. 2. Effect of nisln upon the cell-multiplication of Lactobacillus lactis an(] Streptococcus

lactis.

INHIBITORY EFFECTS OF NISIN

831

TABLE 2 M i n i m u m levels of penicillin, a u r e o m y c i n , s t r e p t o m y c i n , e r y t h r o m y c i n , i n h i b i t i n g m i l k c o a g u l a t i o n b y v a r i o u s lactic c u l t u r e s " Culture 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. t9.

S. lactis U ~ S. lactis C S. lactis F~ S. thermophilus/7 S. thermophilus X S. thermophilus Y S. ther,mophilus Z S. cremoris L. bulgarieus C~ L. bulgaricus b L . lactis Mixed culture

an4

nisin for

Penicillin

Aureomyein

Streptomycin

Erythromycin

Nisin

(~/ml) 0.10 0.10 0.05 0.05 0.10 0.39 0.10 0.15 0.25 0.20 0,10 0.15

(~g/ml) 0.59 0.5,0 0.75 0.10 0.0~5 0.05 0.2,5 0,.5,0 0.2¢5 0.10 6~.25 0,30

(~g/ml) 0.3 1.5 0.6 0.75 0.15 0.3 0,5 0.75 0.2 0.75 0.75 1.5

(~g/m~) 0.6 0.2 0.5 2.0 5.0 0.8 0.5 0.75 1.0 1.0 1,0 1.25

(~/ml) 24 72 24: 48 100 4~ 4 2 8 50 2 150

N o n e o a g u l a t i o n of t h e milk a t e n d o f 16-hr i n c u b a t i o n p e r i o d w a s t a k e n as t h e criterion o f t h e i n h i b i t i o n of t h e cultures by t h e antibiotics.

TABLE 3 E f f e c t of n i s i n - b r o t h u p o n t h e g r o w t h of Bacillus subtilis a n d Bacillus cereus Culture 1. Bacillus Bacillus 9. Bacillus Bacillus

0 hr

subtilis A T C C 6633 subtilis + n i s i n oereus cereus + n i s l n

5.954 5.929 6.121 6.127

6 hr

16 h r

( L o g o f bacterial count) 7.708 8.827 7.7@0 8.786 8.179 8.997 8.121 9.04,1

TABLE 4 E f f e c t of n i s i n u p o n t h e g r o w t h of d i f f e r e n t s t r a i n s of Staphylococcus

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

S. S. S. S. S. S. S. S. S. S. S.

aureus a,ureus a,~xe~ au,re,~s aureus aureus a,~reus aure,~s aureus aureus aureus

Culture 196 lJN 2'09 l~oth l~ed 139 174 125 26 3 21

Nature Positive Positive Positive Positive Positive Positive Positive Weak Weak Weak N e g a t i v e or very weak

8.638 8.601 8.838 8.860

aureus

Transmission %

Coagulase test No.

24 h r

Time

Control

With nisin

(hr) 0.4- 1.4 0.5- 2.1 0 . 5 - 1.8 0 . 8 - 2.6 0.9-10.8 0.7-12.2 1.1-13.2 0.7-2,0.3 14.4-24.1 1.8-19.4 26.0

92.5 3,0.0 28.0 28.5 22.0 18.0 31.0 32.0 29.5 26.0 24.5

25.0 29.5 26,0 24.0 20.5 25.0 35.0 27.5 52.0 28.5 86.0

832

K NI.

u n i t s / n i l ) had to be present in the medium to inhibit the organisms. E f f e c t o f nisin u p o n B. subtilis and B. cereus. Another phase of the study was to determine the effect of nisin upon B . subtilis A T C C 6633 and B . cereus. V a r y i n g quantities of nisinbroth, r a n g i n g between 1 and 5 % v / v , were added to whey broth tubes inoculated with B . subtilis or B. cereus. The tubes were incubated, and at periodic intervals the total counts of bacilli were determined using whey a g a r as the plating medium. The average results of f o u r to five trials with each organism are presented in Table 3. I t was observed that both the organisms had little or no sensitivity towards the antibiotic. E f f e c t o f nisin u p o n S t a p h . aureus. Gowans et al. (8) have reported that some strains of S t a p h . aureus were v e r y sensitive to nisin, whereas some other strains were relatively insensitive. The virulence of food-poisoning staphylococci has been r e p o r t e d to be directly related to their coagulase activity. A study was conducted, therefore, to investigate the effect of nisin u p o n several strains of S t a p h . aureus v a r y i n g in their coagulase production. The antibiotic sensitivity of the cultures was determined by growing the cultures in whey broth containing 5 units of nisin per milliliter, and the turbidity development was measured and taken as an index of their growth. Their coagulase activity was determined by the standard p l a s m a coagulase test. Eleven strains of S t a p h . aureus were graded according to the r a p i d i t y with which they produced the enzyme coagulase, and are listed in Table 4. Culture No. 1 was the most active coagulase p r o d u c e r and coagulated plasma in 25 to 80 rain, and the last culture was almost negative, in that it took more than 24 hr, or in some trials it did not coagulate the plasma. W h e n tested f o r their sensitivity to nisin, it was observed that out of the 11 cultures only two cultures, no. 9 and 11, were sensitive to the antibiotic. B o t h of these sensitive cultures were found to be either weak or negative coagulase producers. The results indicate that nisin might be active against only the coagulase-negative or weak coagulase-producing strains of S t a p h . aureus. I-Iowcver, f u r t h e r study is required to arrive at any definite conclusions. ACKNOWLEDG1VIENTS

Grateful acknowledgment is made to Drs. H. B. Hawley (England), R. Chevalier (France), a~d

SHAHANI

S. Windler (Austria) for providing samples of pure nisin and cultures used in this study. REFERENCES (1) BAKBEft¢,1~. S., BRAU])I~, R., AN]) Hmsctt, A.

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

(6)

(7)

(8)

(9) (10) (11)

(12)

Growth of Pigs Given Skim Milk Soured with NisimProducing Streptococci. nature, 269: 200. 1952. BEXr~ZDOZ, N. J. Preparation of the Antibiotic Nisin. Biochem. J., 45: 486. 1949. BBm~n)(~, N. 5. Counter-current Distribution of Nisins. Nature, 169:707. 1952. BF,n~rDG~, N. J., N~WTON, G. F., A~zI) ABRAH)~, E. P. Purification and Nature of the Antibiotic ~isin. Biochem. J., 52 : 529. 1952. CHEVALI~I¢, R., FOUltNAUD,, J., AND MOCQUOT, G. Nouvelle Technique de Detection des Streptoeoques Inhibiteurs ou Stimulants dans le Lait. Proc. 14th Intern. Dairy Congr., Vo]. 3, 44. 1956. FRAZE~, A. C., AND HICKNKAN, R. Biological Effects of Feeding Nisin and Nisin-Cheese. (Mimeo. Rept.) Research Div. Alpin Barrett Ltd. Co., England. 1956. GAL]~SLOO%T. E. Invloed Van Nisln op die Bacterien Welke Betrokken Zijn of Kunnen Zijn bij Bacteriologische Processen in Kaas en Smeltkaas. Ned. Melk Zuiveltijdshr., 11: 58. 1957. GOWANS, J. L., SMITt~, N., AN]) FLORYJY, ]-I. W. Some Properties of Nisin. Brit. J. Pharmaco]., 7: 4'38. 1952. HAWLEY, H. B. Nisin in Food Technology. Food Manuf., 32: 370. 1957. HmSC'H, A. Some Polypcptide Antibiotics. J. Appl. Bact., 17: 108. 1954.. MAT~'IC~K,A. T. R., am) I-h~scH, A. Further Observations on an Inhibitory Substance (Nisin) from Lactic Streptococci. Lancet, 2 A: 5. 1947. McCLINTOCK, M., SEER, S, L., MAR~OLF, J. J., I'IIRSCH, A., AN]) MOC}QUO~r, G. Action In-

hibitrice des Streptoeoques Producteurs de nisine sur le Developpment des Sporules Anaerobies dans le Formage de Gruyere Fondu. J. Dairy Research, 19: 187. 1952. (13) O'BRIEN, R. T., TierS, D. S., DEIVLIN, K. A., SVUMBO, C. R., Am) LZWlS, J. C. Antibiotics in Food Preservation. I L Studies on the Influence of Subtilin and Nisin on the Thermal Resistance of Food Spoilage Bacteria. Food Technol., 10: 352. 1956. (14) PZTT~, J. W., AN]) KOOY, J. S. The Inhibition of Butyric Acid Fermentation in Cheese by Antibiotic Producing Starter. Proc. 14th Intern. Dairy Congr., Vol. 3: 1172. 1956. (15) SHAHANI, K. M. Factors Affecting Terramycin Activity in Milk, Broth, Buffer, and Water. J. Dairy Sci., 41:382. 1958.