ized cottage cheese by nisin (37 Ã 102. IU/ml and 2.55 x 103 IU/g, respectively) also was investigated. This bacterium was completely inhibited after 24 h at pH ...
D A I R Y FOODS TECHNICAL NOTES Inhibitory Action of Nisin Against Listeria monocytogenes NOREDDINE BENKERROUM and WILLIAM E. SANDINE Department of Microbiology Oregon State University Corvallis 97331-3804 A BST R ACT
The sensitivity of nine strains of Listeria to nisin was determined as well as the minimum inhibitory concentration of nisin necessary to completely inhibit growth of these strains. All strains tested were variably sensitive to nisin and different MIC values were obtained, ranging from 740 to l 0 s IU/ml in trypticase soy agar and from 1.85 to 103 IU/ml in MRS agar. The inhibition of L. monocytogenes ATCC 7644 in TSB trypticase at different pH values and in sterilized and nonsterilized cottage cheese by nisin (37 × 102 IU/ml and 2.55 x 103 IU/g, respectively) also was investigated. This bacterium was completely inhibited after 24 h at pH 5.0 and above. At pH 4.5, 4.0, and 3.5, it was inhibited within 24 h. In cottage cheese no Listeria survivors were found at 24 h at 37 and 4°C whether or not the cheese had been sterilized when as many as .35 x 106 cell/g were added at zero time. INTRODUCTION
Listeria monocytogenes is a foodborne pathogen, which recently has been associated with foodborne disease outbreaks (11, 28). Raw milk has been reported to be a vehicle for this pathogen (14, 19, 20, 23, 27), which can survive the processing of some dairy products (5, 7, 14, 26) or contaminate the plant equipment and thereafter cause postpasteurization contamination.
Received December 28, 1987. Accepted July 11, 1988. 1Paper Number 8373, Oregon Agricultural Experiment Station and Western States Dairy Foods Research Center. 1988 J Dairy Sci 71:3237-3245
Although Bradshaw et al. (2) showed L. monocytogenes would not survive pasteurization, this microorganism has been isolated from pasteurized milk (11, 12), suggesting postpasteurization contamination or improper pasteurization. However, Doyle et al. (6) showed that this organism survives HTST pasteurization of milk (71.7°C for 15 s). It has also been shown that L. monocytogenes survives the manufacture of cottage (5), Camembert (5, 26), and Cheddar (5, 25) cheese as well as NDM (7). Therefore, Listeria may be present in dairy products as a result of postpasteurization contamination or survival during manufacture; growth of this bacterium can occur under temperature abuse but also under refrigeration conditions. In this regard, Rosenow and Marth (24) showed that L. monocytogenes grows well at 4°C in skim, whole, and chocolate milk and in whipping cream and over a temperature range from 0 to 43°C. Although many nonspore-forming bacteria are sensitive to nisin, industrial use of this antibiotic is limited to the prevention of spore outgrowth in processed cheeses and canned foods (8, 13). The earliest use was to prevent the late gas defect in Swiss-type cheeses caused by clostridia (13, 18). It also has been used alone or with subtilin in canned foods to prevent the outgrowth of bacterial spores (13). Recently, Taylor (32) showed that nisin inhibits the outgrowth of Clostridiurn botulinum spores or impedes toxin production by this microorganism. In addition, Ogden (21) and Ogden and Tubb (22) proposed the use of nisin in brewing against Lactobacillus and Pediococcus, the main organism associated with beer spoilage. In the United States, nisin has been placed on the GRAS list and is approved for use in pasteurized cheese spreads (9). The action of nisin is pH-dependent; it is more effective in low pH systems (5, 20). It is stable at pH 2, where it can resist boiling for 10 min, but it is readily destroyed at pH 11 3237
3238
BENKERROUM AND SANDINE
(16, 18). Campbell and Sniff (3) showed that 200 IU/ml are enough to inhibit Bacillus coagulans at pH 5.3 but 560 IU/ml failed to inhibit this bacterium at pH 7.2. According to Henning et al. (15), nisin should be used only in foods where the pH is below 7.0 "to ensure sufficient solubility and stability during processing and storage." As for the safety of nisin, studies done in USSR, Japan, and England (8, 18) have established its nontoxicity. A joint FAO/WHO committee on food additives stated in 1968 that 3,300,000 IU/kg of body weight does not present any undesirable effects, and consequently, they recognize it as a safe food preservative (8). Recent public health concerns about Listeria in food products and the fact that it is a grampositive bacterium prompted us to investigate its sensitivity to nisin. MATERIALS AND METHODS Cultures
Nine strains of Listeria spp. were tested for their sensitivity to nisin. The nine strains also were used to determine the m i n i m u m inhibitory concentration (MIC) of nisin. They included eight strains isolated from clinical cases and a ninth strain (7644) obtained from the American Type Culture Collection (ATCC), Rockville, MD 20852. The clinical isolates were L. monocytogenes 7644K; L. monocytogenes V7 type la; L. monocytogenes 35152; L. monocytogenes Scott A type 4b, L. monocytogenes 15313; L. ivanovii KC 1714 type 5;L. ivanovii C194 type b, and L. saligery LA15. All strains were grown on slants of trypticase soy agar obtained from Baltimore Biological Laboratory (BBL), Baltimore, MD and stored at 4°C. Before each use they were transferred to 10 ml of trypticase soy broth [(TSB: BactoTryptone, Difco Laboratories, Detroit, MI), 15 g; Bacto-Soytone (BBL), 5 g; NaC1, 5 g; and distilled water, 100 ml] and incubated 16 h at 37°C. Antibiotics
Nisin was purchased from Aplin and Barrett Ltd., Trowbridge, England. Its potency was 37 × 106 IU/g. The stock solution was prepared by dissolving .1 g of nisin in 100 ml of distilled water; it was then filter sterilized Journal of Dairy Science Vol. 71, No. 12, 1988
(.22 gtm, Millipore Filter Co., Bedford, MA) and stored at - 2 0 ° C . Sensitivity Testing
A modification of the well assay technique described by Fowler et al. (10) was used: melted TSA was cooled to about 46°C and inoculated with 1% overnight culture of Listeria. Twenty milliliters then were poured into petri dishes and allowed to harden. Wells (8 mm diameter) were then cut into the dishes and filled with 70 /~1 of nisin solution (1 mg/mt). These cultures were incubated at 37°C for 24 to 48 h until inhibition zones were evident. The inhibition zone diameters were then measured with the aid of a graduated ruler.
Determinations of M inimum | nhibitory Concentration
The MIC determinations were done by the agar incorporation method described by Hogg et al. (17). An overnight Listeria culture (25 gtl or approximately 104 cells) was used as inoculum and dispensed as a drop on TSA or MRS agar plates (pH 6.8) containing a given concentration of nisin. Inoculated plates were incubated at 37°C for 24 h. Nisin-free plates (controls) were inoculated in the same way. The concentration was considered to be inhibitory when no growth could be seen after 48 h. Effect of Nisin on Listeria monocytogenes ATCC 7644
The medium used was TSB. Three series of test tubes containing 9 ml each were used. The tubes of each series were adjusted to different pH (7.0, 6.5, 5.5, 5.0, 4.5, 4.0, and 3.5) with 85% lactic acid. To the first series (test), 1 ml of nisin solution (37 × 103 IU/ml) and .1 ml of .6 × 10 s cfu/ml L. monocytogenes ATCC 7644 overnight cultures were added. The final concentrations of nisin and the microorganism in each tube were then 37 × 102 IU/ml (1 mg = 37 × 103 IU) and 6 × 102 cell/ml. To the second series only Listeria was added for the same concentration, i.e., 6 × 102 cell/ml. The third series was a negative control to test the sterility of the medium; neither nisin nor the microorganism was added. Cultures were incubated at 37°C. Listeria count was determined by plate count on TSA at 0, 1, 2, 4, 19, and 24 d.
DAIRY FOODS TECHINCAL NOTE Effect of Nisin on Listeria rnonocytogenes in Cottage Cheese
This experiment was done on sterilized and nonsterilized cottage cheese incubated at 37 or 4°C. Two trials were conducted. The first one was two series of three samples containing 250 g of cheese and 50 ml of pasteurized cream (12% milk fat) each. The samples were thoroughly mixed and sterilized for 20 min at 121°C. Nisin and L. m o n o c y t o g e n e s ATCC 7644 overnight cultures were added to one sample of each series to a final concentration of 2.55 × 103 IU/g and 3.5 × 10 s cell/g, respectively (test). To another sample, the positive control, the culture only was added to the same final concentration. The third sample was a negative control and remained uninoculated and without nisin addition. All these samples were mixed aseptically in a stomacker (Tekmar) for approximately 50 s and replaced in sterile 1000-ml flasks. One series was incubated at 4°C, the other at 37°C. They were then sampled for counting L. m o n o c y t o g e n e s on listeria selective isolation agar (LSI) at 0, 1, 2, 5, 9, 14, 24, and 30 d. LSI (18) consists of: tryptic soy agar (Difco) 45 g with yeast extract (Difco) 5 g; bromcresol purple (Difco) .04 g; and esculin (Sigma Chemical Co., St. Louis, MO) 5 g supplemented with filter sterilized acriflavine hydrochloride (Sigma) and nalidixic acid (Sigma) in solution in .1 N NaOH to a final concentration of .010 and .040 mg/ml, respectively, and a filter-sterilized aqueous solution of polymixin B sulfate (Sigma) to a final concen-
3239
tration of 16 IU/ml. When no Listeria was found in a 1-ml sample by the plate count method, we used the F D A method for Listeria isolation described by Lovett et al. (19). A 25-ml sample was enriched in 225 ml of a selective enrichment medium described by Bannerma and Billie (1) and incubated at 30°C. At 1 d and 7 d, a 10-gtl loop from the enriched culture was streaked on LSI agar and incubated for 24 to 48 h. In the second trial three samples also were prepared (test, positive control, and negative control) in the same way as in the first case except that they were not sterilized. Nisin concentration in the test and the initial concentration of Listeria in the test and the positive control were also the same. The nonsterilized cottage cheese was incubated at 4°C and sampled for plate count on LSI agar at 0, 1, 2, 5, 9, 14, 24, and 30 d. RESULTS A N D DISCUSSION Susceptibility Testing
The well assay for susceptibility test showed that Listeria is inhibited by nisin. The mean of the inhibition zone diameters for each strain tested is shown in Table I. All strains were inhibited by nisin, but important strain differences were observed in the degree of inhibition showing that some Listeria strains are more sensitive to nisin than others: Listeria m o n o c y togenes ATCC 7644 and L. inanovii KC 1714 type 5 were the most sensitive (18 m m diameter) while L. m o n o c y t o g e n e s V7 type la was most resistant (10 mm).
TABLE 1. Diameters of inhibition zones (mm) of Listeria spp. caused by nisin (70 #1 added to assay well). Diameter of inhibition zones, mm ~ Strain L. L. L. L. L. L. L. L. L.
monocytogenes 7644K mon ocytogenes 35152 monocytogenes V7 type la monocytogenes Scott A type 4b ivanovii C194 type b m onocytogenes 15313 saligery LA15 ivanovii KC1714 type 5 monocytogenes ATCC 7644
Nisin
SD
12 11 10 11 11 11 12 18 18
.71 .41 .90 .73 .16 .16 1.08 1.41 1.47
1Mean values of four determinations. Journal of Dairy Science Vol. 71, No. 12, 1988
3240
BENKERROUM AND SANDINE
Determ ination of Minimum Inhibitory Concentration
The MIC ranges of the nine strains tested are shown in Table 2. These results show again that L. m o n o c y t o g e n e s ATCC 7644 is the most vulnerable strain to nisin among those tested. It has the lowest MIC (740 IU/ml), followed by L. ivanovii KC1714 type 5. Listeria m o n o c y t o g e n e s V7 type la had the highest MIC (1.18 X l 0 s IU/ml). In general, the MIC ranges obtained were relatively high and subject to variations depending on the assay conditions, mainly pH and composition of the medium. The pH of TSA used in this experiment was 7.3, which is not optimum for nisin action, as stated earlier. Also, MIC ranges obtained on MRS lactobacilli agar (Difco) were much lower than those obtained on TSA (Table 2). One explanation for this is the lower pH of MRS (6.8), which may be more suitable for nisin action than pH 7.3. Also, MRS contains some components, which may synergistically inhibit Listeria with nisin increasing the overall effect. Sodium acetate contained in this medium is indeed inhibitory to a number of gram-positive and gram-negative bacteria. These results suggest that one must be careful when wanting to minimize nisin addition to products based on the MIC determinations done under specific conditions. Growth of Listeria monocytogene$ A T C C 7466 at Different pH in Presence or Absence of Nisin
Growth of L. m o n o c y t o g e n e s in TSB at different pH in the presence and absence of nisin is shown in Figure 1. Listeria m o n o c y t o g e n e s
grew well without nisin at pH of 5.5 to 7.0. At pH 5, growth was slow but not completely inhibited. After 24 d no Listeria were found in 1-ml samples; however, regrowth (6.3 × 102 cell/ml) was observed by the 19th d at this pH. The inhibitory effect of pH became clear at pH 4.5 and below. The pathogen was eliminated from the sample after 48 h at pH 4.5 and after only 24 h at pH 3.5. No regrowth was observed at pH lower than 5.0. Similar results were reported by Conner et al. (4) who showed that L. m o n o c y t o g e n e s was completely inhibited below pH 4.6 b u t grew well at pH 5.6. The acid used to adjust the pH was also lactic acid. Figure 1 shows also the growth of L. m o n o c y t o g e n e s ATCC 7466 in TSB with 37 x 102 IU/ml of nisin. No survivors were found after 24 h in a 1-ml sample at all pH. At pH 7.0 and 6.5 an increase of Listeria was observed in the first 24 h to about 104 cfu/ml and then no Liseria was detected in 1 ml within the next 24 h. At pH 4.5, 4.0, and 3.5 the cells were killed within 24 h. These data show that the bacterium is more vulnerable to nisin at low pH, either because nisin is more effective at low pH, as it has been shown earlier (3, 15), or because of an additive effect of acidity and nisin action on Listeria. However, unlike the report of Henning et al. (15), nisin was still effective against Listeria at pH 7.0. According to these authors nisin is less soluble at pH 7.0, which impeded its effectiveness. Hurst (17), however, reported that a high protein concentration enhances the solubilization of nisin. According to Eapen et al. (8), the solubility of nisin in water at pH 7.0 is 75 /ag/ml (the equi-
TABLE 2. Minimum inhibitory concentrations (MIC) (pH 7.3) of nisin on Listeria monocytogenes ATCC 7644 determined on trypticase soy agar (TSA) and MRS agar at (pH 6.8). MIC (IU/ml) Strain
L.. monocytogenes 7644K L. monocytogenes 35152 L. monocytogenes V7 type la L. monocytogenes Scott A type 4b L. ivanovii C194 type b L. monocytogenes 15313 L. saligery LA15 L. ivanovii KC 1714 type 5 L. monocytogenes ATCC 7644
Journal of Dairy Science Vol. 71, No. 12, 1988
TSA 1.48 X 104 1.48 X 104 1.18 X 10 s 1.18X 104 74 X 103 1.48 × 104 1.48 X 104 14.8 X 102 740
MRS 37 3.7 3.36 X 103 1.65 X 103 37 1.65 X 103 37
3.7 1.85
DAIRY FOODS TECHNICAL NOTE
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3241
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Days Figure 1. Effect of pH and added nisin (37 X 102 IU/ml) on growth of Listeria monocytogenes in trypticase soy broth incubated at 37°C.
valent of 2.8 x 103 IU/ml), which is sufficient
to inhibit all microorganisms susceptible to this antibiotic. An average of 2 to 10 units of nisin/ ml are enough to inhibit a cell concentration of 2 x 10S/ml (16). Taylor (32) showed that 50 to 200/ag/g (1.9 x 10 a to 7.4 x 103 IU/g) were necessary to completely inhibit C. botulinum in various canned foods. Also, Mohamed et al. (20) showed that only 32 IU/ml are necessary to inhibit L. monocytogenes 4379 at pH 7.4 and 37°C. They also showed that the sensitivity of this strain decreases when the temperature of incubation decreases: 256 IU/
ml are required to inhibit completely L. m o n o cytogenes 4379 growth at 22°C and pH 7.4.
However, this amount is 16-fold reduced at pH 5.5 at the same temperature. Furthermore, in dairy products, the solubility of nisin does not seem to be a concern, because most have a low pH and they contain enough protein to help solubilize nisin. Furthermore, nisin has already been used successfully in cheeses, as
mentioned earlier. From Figure 1 it may be seen that in the positive controls (samples without nisin) at pH 5.5 to 7.0, the number of Listeria keeps increasing, whereas in the test samples (plus nisin) Listeria substantially decline after 24 h, indicating an important bactericidal effect of this antibiotic. Journal of Dairy Science Vol. 71, No. 12, 1988
3242
B E N K E R R O U M A N D SANDINE
Effect of Nisin on Liseteria monocytogenes A T C C 7644 in Sterilized and Nonsterilized Cottage Cheese
Figure 2 shows the growth curves of the pathogen in the positive control and the test samples of the sterilized cottage cheese at 4 and 37°C. No Listeria could be found in 1-mI
of sample after 1 d at 4°C as well as at 37°C. Furthermore, the pathogen could not be recovered by the FDA method for Listeria isolation either after 1 d nor after 1 wk, which confirms that it was killed by nisin, not only inhibited or injured. The same figure shows also that Listeria grows well in sterilized cottage cheese
10
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Days Figure 2. Growth of Listeria monoeytogenes 7644 at 4 and 37°C in the presence and absence of nisin (2.55 × 103 IU/g) in sterilized cottage cheese. Journal of Dairy Science Vol. 71, No. 12, 1988
DAIRY FOODS TECHINCAL NOTE (positive sample) despite its low pH, which was in our case 4.9 to 5.0, while the synthetic medium showed that the pathogen was greatly although not completely inhibited (regrowth was observed in the 19th d) (Figure 2) at pH 5. This may be explained by the richness of cottage cheese that provides Listeria with some growth factors not found in the synthetic medium, or by the protective effect that cheese proteins may have for the pathogen as it has been shown with Clostridium botulinum, which can grow in canned foods at lower pH than in synthetic media (31). It may be a combination of these two factors as well. Table 3 summarizes the effect of nisin on Listeria in nonsterilized cottage cheese at 4°C. It appears that the cheese starter bacteria are not inhibited by LSI medium. However, the latter form larger colonies and ferment the esculin more slowly when used as a fermentable carbohydrate in the medium. Some suspicious colonies (smaller and turn yellow f a s t e r ) w e r e selected and Gram stained. Such colonies were gram-positive cocci and therefore assumed to be starter bacteria. The pathogen was not frequently found in the positive control either; one or two colonies, at the most out of 10 were Listeria, sometimes none was Listeria. This suggests that starter bacteria exhibit an antagonistic effect against Listeria, which is no surprise; in fact, the inhibitory effect of lactic acid bacteria on pathogens and spoilage microorganisms is now well known. Nonetheless, the
3243
inhibition of the pathogen by the starter bacteria is no guarantee of the safety of this cheese. Some cells can survive as it was found here and elsewhere (4, 27, 30). However, the addition of nisin not only inhibits the growth of Listeria but also kills the bacterium. The antibiotic is therefore able to protect this product from Listeria when contaminated either from the raw milk or as a result of postpasteurization contamination. Most importantly, nisin conserves its effectiveness at a low temperature for a long time (13), ensuring protection of the product against Listeria contamination when it is stored under refrigeration. Furthermore, nisin seems to delay the growth of other spoilage psychrotrophs that can exist or contaminate cottage cheese. In effect, in this experiment, nonsterilized cottage cheese samples without nisin spoiled 1 wk earlier than those containing the antibiotic (the spoilage was judged by the alteration of the physical appearance and the smell). The counts are also lower in the test than in the control samples (Table 3). This work shows that in addition to the classical use of nisin to prevent outgrowth of bacterial spores in foods, it can also be used to prevent growth of some foodborne pathogens of major concern such as L. monocytogenes. The growth of this bacterium that can occur at a relatively low pH (pH 4.9) and under refrigeration can be overcome by nisin addition. The sensitivity of Listeria to nisin was shown to be somewhat strain dependent which should be
TABLE 3. Influence of nisin on microbial counts, which developed on Listeria selective isolation medium in nonsterilized cottage cheese contaminated with Listeria monocytogenes ATCC 7644 during incubation at 4°C. Days Treatment
0
1
2
5
9
14
24
30
Nisin added ~'2
.04
.08
.3
.2
4.0
.2
.1
N o nisin 1,3
.04
.1
.8
.6
3.0
3.0
2.0
3
Control 4
. . . .
.9
.5
4.0
3
(cfu/ml X 107)
09
.06
.02
.1
15 × 10 s cell/g of L. monocytogenes added. 2Although while starter bacteria present in the cottage cheese grew on LSI medium, L. monocytogenes could not be found on plates of sample from the cheese containing nisin. 3Colonies were cheese starter bacteria and L. monocytogenes. 4No L. monocytogenes added, so colonies were cheese starter bacteria. Journal of Dairy Science Vol. 71, No. 12, 1988
3 244
B E N K E R R O U M A N D SANDINE
t a k e n i n t o a c c o u n t in d e t e r m i n i n g a m o u n t s t o b e a d d e d in o r d e r t o o f f e r a s a f e t y f a c t o r . A l s o , s i n c e n i s i n is h a r m l e s s f o r h u m a n s , its u s e a s a f o o d a d d i t i v e in t h e U n i t e d S t a t e s t o i n h i b i t pathogens warrants further consideration. ACKNOWLEDGMENTS G i f t o f e i g h t s t r a i n s o f Listeria f r o m J. K . K o n d o , U t a h S t a t e U n i v e r s i t y , is a c k n o w l e d g e d . T h e r e s e a r c h w a s s u p p o r t e d in p a r t b y a g r a n t from the Western States Dairy Foods Research Center. REFERENCES 1 Bannerma, E. S., and J. Bitlie. 1988. A new selective m e d i u m for Listerm sl0p. from heavily c o n t a m inated material. AppI. Environ. Microbiol. 54:165. 2 Bradshaw, J. G., J. T. Peeler, J. J. Corwin, J. M. Hunt, and R.M. Twedt. 1987. Thermal resistance of Listem monocytogenes in dairy products. J. Food Prot. 50:543. 3 Campbell, L. L., and E. E. Sniff. 1959. Nisin sensitivity of Bacillus coagulans. AppI. Microbiol. 7: 289. 4 Conner, D. E., R. E. Brackett, and L. R. Beuchat. 1986. Effect of temperature, sodium chloride and pH on growth o f Listerm monoeytogenes in cabbage juice. AppI. Environ. Microbiol. 52:59. 5 Doyle, M. P. 1987. Survival characteristics of Listeria monocytogenes during processing. Page 198 in Abstr. Inst. Food Technol. Mtg., June 1 6 19. 6 Doyle, M. P., K. A. Glass, J. T.Berry, G. A. Garcia, D. J. Pollard, and R. D. Schultz. 1987. Survival of Listerm monocytogenes in milk during high-temperature, short-time pasteurization. Appl. Environ. Microbiol. 53:1433. 7 Doyle, M. P., L. M. Meske, and E. H. Marth. 1985. Survival of Listeria monocytogenes during the m a n u f a c t u r e and storage of n o n f a t dry milk. J. Food. Prot. 48:740. 8 Eapen, K. C., R. Sankaran, and P. K. Vijaraghavan. 1983. The present status on the u s e of nisin in processed foods. J. Food Sci. Technol. 20:231. 9 Federal Register. 1988. Nisin preparation; affirmation of GRAS status as a direct h u m a n food ingredient. Vol. 53(66):11247. 10 Fowler, G. G., B. Jarvis, and J. Trainer. 1975. The assay of nisin in foods. R. G. Board and D. W. Lovelock, ed. Some m e t h o d s for microbiological assay. Academic Press, New York, NY. 11 Flemming, D. W., S. L. Cochi, K. L. MacDonald, J. B. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Homles, A. Audurier, C. V. Broom, and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New England J. Med. 312:404. 12 Garayza'bal, J.F.F., L. D. Rodriguez, and Boland, J.L.B. Cancelo, and G. S. Fernfindez. 1986. Listeria monocytogenes clans le lait pasteurisE. Can J. Microbiol. 32:149.
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13 Gibbs, B. M., and A. Hurst. 1964. Limitations of nisin as a preservative in non dairy foods. 4th Int. Symp. Food Microbiol., SIK, Goteborg, Swed. 14 Hayes, P. S., J. C. Freeiey, L. M. Graves, G. W. Ajello, and D. W. Flemming. 1986. Isolation of Listeria monocytogenes from raw milk. AppI. Environ. Microbiol. 51:438. 15 Henning, S., R. Metz, and W. P. Hammes. 1986. New aspects for the applicationof nisin to food products based on its m o d e of action. Int. J. Food. Microbiol. 3:135. 16 Hirsch, A. 1950. The assay of the antibiotic nisin. J. Gen. Microbiol. 4:70. 17 Hogg, G. M., M. F. Patterson, and J. G. Barr. 1987. Correlation between antibiotic sensitivity testing by conventional and conductivity measurements. J. AppI. Bacteriol. 62:189. 18 Hurst, A. 1981. Nisin: a review. Advances in microbiology. Vol. 27. Academic Press, New York, NY. 19 Lovett, J., D. W. Francis, and J. M. Hunt. 1987. Listeria monocytogenes in raw milk: detection, incidence and pathogenicity. J. Food. Prot. 50:188. 20 Mohamed, G. E., A. Seaman, and M. Woodbine. 1984. F o o d antibiotic nisin comparative effect on Erysipelotbrlx and Listeria. Proc. 4th Int. Syrup. Antibiot. Agric., March 27 to 31, 1983 and the University of Nottingham. Blackwell Publ., Ltd., London, Engl. 21 Ogden, K. 1986. Nisin: a bacteriocin with a potential use in brewing. J. Inst. Brewing 22:379. 22 Ogden, K., and R. S. Rubb. 1985. Inhibition of beer spoilage lactic acid bacteria by nisin. J. Inst. Brewing 91:390. 23 Rodriguez, D., J.F.F. Garayzabal, J.A.V. Boland, and E. R. Fernandez. 1985. Isolation de microorganisms du genre Listeria a partir de lait cru destind a la c o n s o m m a t i o n h u m a i n e . Can. J. Mierobiol. 31:939. 24 Rosenow, E. E., and E. H. Marth. 1987. Growth of ListeHa monocytogenes in skim milk, whole and chocolate milk and whipping cream during incubation at 4, 8, 13, 21, and 35°C. J. Food Prot. 50: 542. 25 Ryser, E. T., and E. H. Marth. 1987. Behavior of Listeria monocytogenes during the m a n u f a c t u r e and ripening of cheddar cheese. J. Food Prot. 50: 7. 26 Ryser, E. T., and E. H. Marth. 1987. Fate of Listeria monocytogenes during the m a n u f a c t u r e and ripening of C a m e m b e r t cheese. J. Food Prot. 50:372. 27 Reyser, E. T., E. H. Marth, and M. P. Doyle. 1985. Survival of Listeria monocytogenes during m a n u facture and storage o f cottage cheese. J. Food Prot. 48: 746. 28 Schlech, W. F., P. M. Lavinge, R. A. Bortolussi, A. C. Allen, E. V. Haldone, A. J. Wort, A. W. Hightower, S. E. Johnson, S. H. King, E. S. Nicholls, and C. V. Broome. 1983. Epidemic listeriosis evidence for transmission by food. New England J. Med. 308:203. 29 Schultz, G. 1967. Studies on occurrence of Listeria in raw milk. Montash. Veterinaermed. 22:766. 30 Siragusa, G. R., and M. G. Johnson. 1987. An agar
DAIRY FOODS TECHNICAL NOTE plating medium for isolation of Listeria monocytogenes inoculated in yogurt. Page 106 in Abstr. Inst. Food Techol. Mtg., June 16-19. 31 Tanaka, N. 1982. Toxin production by Clostridium
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botulinum in media at pH lower than 4.6. J. Food Prot. 45:234. 32 Taylor, S. L. 1986. Nisin as an antibotulinal agent for food products. US Pat. No. 4,598,972.
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