Utilization of cheddar cheese containing nisin as an antimicrobial agent in other foods

ELSEVIER

International Journal of Food Microbiology 24 (1994) 227-238

International Journal of Food Microbiology

Utilization of cheddar cheese containing nisin as an antimicrobial agent in other foods Edmund A. Zottola *, Tom L. Yezzi, Diran B. Ajao, Robert F. Roberts 1 Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St. Paul, MN 55108, USA

Abstract Cheddar cheese made with nisin-producing lactococci contained between 400 and 1200 IU of nisin per gram of cheese. Cultures used were Lactococcus lactis ssp. cremoris JS102, a nisin-producing transconjugant developed in the laboratories of Dr. L.L. McKay and Lactococcus lactis ssp. lactis N C D O 1404 obtained from the National Collection of Food Bacteria, Reading, England. Pasteurized process cheese spreads with 53% and 60% moisture and 0, 301 and 387 IU n is i n / g were manufactured and inoculated with 2000 spores of Clostridium sporogenes PA 3679 during manufacture. The heat process did not reduce nisin activity in the cheese spreads. The spreads were incubated at 22° and 37°C for 90 days. Spoilage was detected by the presence of gas a n d / o r odor in the packages. The shelf-life of the nisin-containing cheese spreads was significantly greater than that of the control cheese spreads at the lower temperature at both moisture levels, whereas the keeping quality of the higher moisture cheeses at the higher temperature was not significantly different. Club cheese or cold pack cheese spreads with moisture levels of 44% and 60% and 0, 100 and 300 IU n i s i n / g were made. These cold processed cheese spreads were inoculated with 1000 cfu per g of Listeria monocytogenes V7, Staphylococcus aureus 196E and spores of C. sporogenes PA 3679. Heat shocked spores of PA 3769 at the same number were added to separate lots of the cheese spread. The cold pack cheese spreads were incubated at 23 ° and 37°C for up

Published as Paper No. 20743 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under Project 18-56, supported by Hatch Funds and funds from the Minnesota-South Dakota Dairy Foods Research Center. * Corresponding author. i Present address: Department of Food Science, Pennsylvania State University, University Park, PA 16801, USA. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1605(94)00127-8

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E.A. Zottola et al. / InternationalJournal of Food Microbiology24 (1994) 227-238

to 8 weeks. Samples were taken weekly and analyzed for surviving organisms. Significant reductions in numbers of the non-sporeforming test microbes were noted at both temperatures, at both moisture levels and both levels of nisin. Heat shocking the spores was needed to show reduction in numbers during the storage of the cold pack cheese spreads. The data obtained in this study suggest that the use of nisin-containing cheese as an ingredient in pasteurized process cheese or cold pack cheese spreads could be an effective method of controlling the growth of undesirable microorganisms in these processed foods.

Keywords: Nisin; Cheddar cheese; Pasteurized process cheese spread; Cold-pack cheese spread; Antimicrobial

1. Introduction

Bacteriocins are defined as inhibitory substances produced by certain genera of bacteria that are able to inhibit the growth of other similar species or genera of bacteria. The bacteriocin that has received the most interest is nisin, the one first characterized by Mattick and Hirsch in 1944. This inhibitory substance is produced by certain strains of Lactococcus lactis ssp. lactis and has been shown to have an inhibitory effect on the growth of a number of Gram-positive organisms including species in the genera Clostridia, Bacillus, Lactobacillus, Lactococcus, Micrococcus, Listeria, Pediococcus, Staphylococcus, Streptococcus and Erysipelothrix (Benkerroum and Sandine, 1988; Carminati, et al.; 1989, Chung et al., 1989; Harris et al., 1989; Hurst, 1981; Lipinski, 1977; M o h a m e d et al., 1981). A significant amount of research has been done on this bacteriocin to characterize it and its potential for commercial application (Hurst, 1981; Lipinska, 1973). Nisin has been shown to be a polypeptide of 34 amino acids with an apparent molecular weight of 3500 Da (Gross and Morell, 1971). One of the first to report the use of nisin-producing cultures of lactococci to inhibit the outgrowth of spoilage organisms in processed cheese was McClintock et al. (1952). Milk cultured with a nisin-producing culture was added to processed Gruyere cheese and a significant increase in shelf-life was observed. Other investigators have utilized purified nisin in the form of Nisaplin TM, available from Aplin and Barrett, Ltd., Trowbridge, London, England as an additive to process cheese to inhibit outgrowth of spoilage and potentially pathogenic organisms (Berridge, 1953; Scott and Taylor, 1981; Somers and Taylor, 1987; Tanaka et al., 1986). Nisin has been used in other foods as well, primarily as an adjunct preservative to reduce the heat process necessary to destroy anaerobic sporeforming bacteria (Fowler, 1979). Adoption of the use of nisin in food preservation in the United States has not been as wide spread as in Europe. A novel approach to the use of bacteriocins to control undesirable microbes in food would be to produce a fermented food that would naturally contain significant quantities of a bacteriocin. This product could then be utilized as an ingredient containing a natural microbial inhibitor in other foods to control both spoilage and potentially pathogenic bacteria.

E.A. Zottola et al. / International Journal of Food Microbiology 24 (1994) 227-238

229

The objectives of the study reported here were to develop Cheddar cheese containing significant quantities of nisin and to utilize the nisin-containing cheese as an ingredient in other foods to inhibit the growth of undesirable microbes.

2. Materials and methods

Cultures. The cultures used in this study are described in Table 1. All cultures were maintained in frozen storage by first incubating in appropriate media at optimum growth temperature for 8 to 10 h. Where needed, the pH was adjusted to 6.5 and the cultures frozen at -50°C. Prior to use, the cultures were removed from frozen storage, thawed and transferred to fresh sterile media and incubated. Cultures were transferred twice prior to use. Spores of C. sporogenes PA 3679 were prepared using the biphasic beef heart medium described by Bruch et al. (1968), modified by Ababouch and Busta (1985) and Roberts and Zottola (1993). p H control. Individual strains of nisin-producing Lactococci were grown in 10% reconstituted skim milk (RSM) with or without pH control of the media. The pH

Table 1 Bacterial cultures and cultural conditions used in this study Culture

Lactococcus lactis ssp. cremoris JS 102 Lactococcus lactis ssp. lactis 1404 Micrococcus luteus 10240

Source

Growth medium

Incubation Temperature (°C)

Time (h)

L.L. McKay

10% RSM a

30

15

NCDO b

10% RSM

30

15

ATCC c

Nisin assay agar f

30

48

NFPA ~

Biphasic beef heart medium g

37

4 weeks

U of MN e

Tryptic soy broth h

30

18

U of MN e

Tryptic soy broth h with 0.6% yeast extract

30

18

Clostridium sporogenes PA 3679

Staphylococcus aureus 196E

Listeria monocytogenes strain V7 type la

a RSM, 10% solids reconstituted skim milk powder, sterile. b NCDO, National Collection of Food Bacteria, Reading, England. c ATCC, American Type Culture Collection, Baltimore, MD, USA. a NFPA, National Food Processors Association, Washington, DC, USA. e U of MN, Cultures from collection in our laboratory. f Nisin Assay Agar, Tramer and Fowler (1968). h Tryptic Soy Broth, Difco, Detroit, MI, USA.

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Table 2 Composition of the cheese and pasteurized cheese spreads used in this study Cheese type

% Solids

% Fat

% Moisture

pH

IU NISIN/G

Cheddar a (control) Cheddar a (nisin) Pasteurized process cheese spread-low b Pasteurized process cheese spread-high c Pasteurized process cheese spread-low b Pasteurized process cheese spread-high c Cheddar d (pH control)

63 63 47 40 47.5 41 .

32 31.5 23 23 23 19 .

37 37 53 60 52.5 59 .

5.3 5.2 -

0 722 0 0 387 301 1170

.

-

Mean of five lots of cheese given. b Low-moisture pasteurized process cheese spread. ~ High-moisture cheese spread. c Cheese made with starter culture grown under pH controlled system.

was m e a s u r e d at h o u r l y intervals using an O r i o n digital ion a n a l y z e r ( O r i o n , C a m b r i d g e , M A ) e q u i p p e d with an O r i o n 9 1 - 0 5 p H e l e c t r o d e . W i t h t h e p H c o n t r o l l e d c u l t u r e s t h e p H was a d j u s t e d to p H 6.5 _+ 0.2 with 5 N N a O H . S a m p l e s w e r e r e m o v e d p e r i o d i c a l l y for nisin assay a n d for d e t e r m i n a t i o n of the n u m b e r of viable o r g a n i s m s using M-17 a g a r (Difco, D e t r o i t , M I ) s u p p l e m e n t e d with 0.5% lactose. P l a t e s w e r e i n c u b a t e d at 30°C for 48 h. Cheddar manufacture. C h e d d a r c h e e s e was m a n u f a c t u r e d using the p r o c e d u r e s d e s c r i b e d by Kosikowski (1982). P a s t e u r i z e d p r o c e s s c h e e s e s p r e a d s w e r e m a n u f a c t u r e d as d e t a i l e d by R o b e r t s a n d Z o t t o l a (1993). Cold p a c k c h e e s e s p r e a d s w e r e m a n u f a c t u r e d following t h e m e t h o d s given by Kosikowski (1982). T h e c o m p o s i t i o n of the v a r i o u s c h e e s e types is given in T a b l e 2. Determination o f bacteriocin content. T h e nisin c o n t e n t of the original cheese, the p a s t e u r i z e d p r o c e s s c h e e s e s p r e a d s a n d the cold p a c k c h e e s e s p r e a d s w e r e q u a n t i fied using t h e well diffusion assay d e s c r i b e d by T r a m e r a n d F o w l e r (1964). T h e m o d i f i c a t i o n s to the assay as s u g g e s t e d by t h e British S t a n d a r d s I n s t i t u t e p u b l i c a tion 4020 w e r e i n c o r p o r a t e d into t h e p r o c e d u r e u s e d (British S t a n d a r d s Institute, 1974). Shelf-life studies. B a t c h e s of p a s t e u r i z e d p r o c e s s c h e e s e with or w i t h o u t a d d e d s p o r e s of C. sporogenes P A 3679 w e r e i n c u b a t e d at 23°C a n d 37°C for 90 days. S p o i l a g e was a p p a r e n t by t h e p r e s e n c e o f gas in the p a c k a g e a n d o r the p r e s e n c e of t h e o d o r a s s o c i a t e d with t h e g r o w t h o f P A 3679. T h e cold p a c k c h e e s e s p r e a d s i n o c u l a t e d with Listeria monocytogenes w e r e s t o r e d at 4°C a n d 23°C. T h o s e i n o c u l a t e d with Staphylococcus aureus w e r e s t o r e d at 23°C w h e r e a s those c o n t a i n i n g C. sporogenes P A 3679 w e r e s t o r e d at 23°C. Sample analysis. S a m p l e s w e r e e v a l u a t e d for n u m b e r s o f specified b a c t e r i a at 0, 3, 7, 14, 21, 28, a n d 56 days as d e s c r i b e d below:

E.A. Zottola et al. / International Journal of Food Microbiology 24 (1994) 227-238

231

E n u m e r a t i o n o f L. monocytogenes and S. aureus. To determine the number of L. monocytogenes and S. aureus in the samples, four grams of cold-pack cheese was

diluted with 36 ml of a 2% solution of sodium citrate. The sample was blended in a Stomacher (Tekmar Company, Cincinnati, OH) for 2 min min and 1 ml was distributed over four agar plates. L. monocytogenes was enumerated using Oxford Listeria agar (Oxoid, Unipath Ltd., Basingstoke, Hampshire, England) and S. aureus was enumerated on Baird-Parker agar (Difco, Detroit, MI). Inoculated plates for both organisms were incubated at 30°C for 48 h and then counted. C. sporogenes enumeration. Samples containing C. sporogenes spores were enumer-

ated in tryptone peptone glucose yeast extract broth (TPGYE) (Difco, Detroit, MI) using the most probable number technique (MPN) as described by Oblinger and Koburger (1986). Ten gram samples of cold-pack cheese containing C. sporogenes spores were diluted in 90 ml of a freshly exhausted 2% solution of sodium citrate. Samples were blended in a Stomacher for 1 min. To activate the spores, samples inoculated with nonheat-shocked spores were then heat-shocked at 85°C for 15 min. Samples containing previously heat shocked spores were diluted in 90 ml of a freshly exhausted 2% solution of sodium citrate and blended in a Stomacher for 1 rain. A three-tube MPN was used. Samples were serially diluted and used to inoculate tubes of TPGYE. Tubes were incubated in anaerobic jars (Gas-Pak, BBL, Baltimore, MD) for 3 days at 37°C. Tubes were observed for turbidity and characteristic odor of C. sporogenes. Numbers were determined using the 3-Tube MPN tables as given by Oblinger and Koburg (1986).

3. Results and discussion

Several strains of nisin-producing lactococci were screened for their ability to produce sufficient acid to successfully manufacture Cheddar cheese (Roberts et al., 1992). Two different strains, Lactococcus lactis ssp. cremoris JS102 (Steele and McKay, 1986) and Lactococcus lactis ssp. lactis 1404, were selected and utilized to manufacture several lots of Cheddar cheese. The composition and pH development during the process was normal. The average composition of the five lots of cheese made is given in Table 2. The average quantity of nisin in the five vats as given in Table 2 was 722 IU of nisin/g (Roberts et al., 1992). The nisin content of the cheese was increased to over 1000 I U / g by utilizing pH control during the growth of the starter culture. The increase in nisin content of the cheese appeared to be related to the increase in cell mass obtained when pH control was used during growth of the cultures used in making the cheese (Yezzi et al., 1993). High moisture (60%) and low moisture (53%) pasteurized process cheese spreads with and without nisin were manufactured. The high moisture cheese spread contained 301 IU nisin/g and the low moisture cheese spread contained 387 IU nisin/g. The composition of the spreads is given in Table 2. The nisin-containing Cheddar cheese was the source of the nisin in the cheese spreads. Non-nisin containing spreads were made from the control cheese. Approximately

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Table 3 Shelf-stability of p a s t e u r i z e d process c h e e s e s p r e a d with and w i t h o u t nisin i n o c u l a t e d with spores of clostridium sporogenes P A 3679 Storage temperature

% MO a

IU N/g

(oc) 22

37

53 53 60 60 53 53 60 60

0 387 0 301 0 387 0 301

Days to spoilage d e t e c t i o n (odor, gas) C o n t r o l (no nisin)

nisin

67 14 24 4 -

< 90 87 < 90 l(/

a % MO, p e r c e n t m o i s t u r e in cheese. I U N / g , I U of nisin p e r g r a m of spread. I n o c u l a t i o n level a p p r o x i m a t e l y 2000 s p o r e s p e r g of spread.

2000 spores of C. sporogenes PA 3679 per gram of cheese spread were added during the heat processing of the spreads and were thus activated by the heat treatment. The eight batches of high and low moisture cheese spread were incubated at 22°C for up to 6 months and at 37°C for 90 days. The data presented here represent the first 90 days of the study. Little, if any, additional spoilage occurred during the increased storage time at the lower temperature. The a w of the cheese spreads was the same regardless of the moisture content and there were no other added inhibitory substances such as those described by Tanaka et al. (1986). The shelf-life of the pasteurized process cheese was a reflection of the nisin content of the spreads. The results of the shelf-life study are given in Table 3. As might be expected, the high moisture cheese spreads spoiled faster than the low moisture cheese spreads. Cheese spreads containing nisin had longer shelf stability than those without nisin when stored under similar conditions. All high moisture cheese spreads stored at 37°C spoiled within 10 days regardless of nisin content. The preservative effect of nisin was much more evident for the same cheese when stored at 22°C. All eight batches of the high moisture cheese spreads without nisin spoiled within 14 days of storage at 22°C, whereas the nisin-containing high moisture cheese spread did not show spoilage until the 87th day at this temperature. The low moisture cheese spreads with or without nisin had a much longer shelf-life than the high moisture spreads (Table 3). At 37°C, the low moisture cheese spread without nisin spoiled within 24 days, whereas the nisin-containing spread did not spoil within the 90-day incubation period. The same was true at 22°C. The nisin-containing spread did not spoil during the 90-day test, in fact it was still good after 6 months. The non-nisin containing spreads spoiled with 67 days at 22°C. These results suggest that using Cheddar cheese containing nisin as

233

E.A. Zottola et al. / International Journal of Food Microbiology 24 (1994) 227-238

Table 4 Formulas of cold pack cheese spreads used in the study Ingredients

High moisture a ( % )

LOWmoisture u (%)

Cheese Dry whey Salt Ice

53 6 1 40

79 5.5 0.5 15

Cold pack cheese spreads containing 0, 100 and 300 IU nisin/g were made with these formula by mixing control cheese (no nisin) with nisin-containing cheese. The pH of all spreads was adjusted to 5.1 with lactic acid. a Finished spread contains 60% moisture. b Finished spread contains 44% moisture.

an a n t i - m i c r o b i a l a g e n t will result in e x t e n d e d shelf-life of p a s t e u r i z e d p r o c e s s cheese spread. T h e p a s t e u r i z e d p r o c e s s c h e e s e s p r e a d f o r m u l a t i o n s u s e d in t h e study w e r e d e s i g n e d to assure m a x i m u m p o t e n t i a l for spoilage. If o t h e r inhibitors h a d b e e n a d d e d as s u g g e s t e d by T a n a k a et al. (1986), it m i g h t have b e e n p o s s i b l e to e l i m i n a t e s p o i l a g e c o m p l e t e l y . It was o u r i n t e n t to show t h e p o t e n t i a l for t h e use o f n i s i n - c o n t a i n i n g c h e e s e as a n a n t i - m i c r o b i a l agent, thus we d e c i d e d not t o a d d o t h e r p o t e n t i a l preservatives. A s e c o n d p r o c e s s e d c h e e s e p r o d u c t s e l e c t e d for e v a l u a t i o n was c o l d - p a c k c h e e s e s p r e a d . This p r o d u c t is m a n u f a c t u r e d by c o l d - b l e n d i n g c h e e s e a n d o t h e r i n g r e d i e n t s to p r o d u c e a s m o o t h , s p r e a d a b l e c h e e s e p r o d u c t with r e a s o n a b l e r e f r i g e r a t e d shelf-life (Kosikowski, 1982). C o l d - p a c k c h e e s e s p r e a d s with a n d w i t h o u t nisin with 44% a n d 60% m o i s t u r e w e r e m a d e by b l e n d i n g n i s i n - c o n t a i n i n g c h e e s e with n o n - n i s i n c o n t a i n i n g c h e e s e a n d o t h e r i n g r e d i e n t s . T h e f o r m u l a s for t h e c h e e s e s p r e a d s a r e given in T a b l e 4. B o t h m o i s t u r e levels of c h e e s e s p r e a d s w e r e f o r m u l a t e d to c o n t a i n 0, 100 a n d 300 I U nisin p e r gram. T h e a m o u n t s o f n i s i n - c o n t a i n i n g c h e e s e a n d n o n - n i s i n c o n t a i n i n g c h e e s e u s e d in t h e s e f o r m u l a t i o n s a r e given in T a b l e 5. T h e f o r m u l a t i o n s w e r e d e s i g n e d to a s s u r e m a x i m u m m i c r o bial g r o w t h to test t h e ability o f t h e n i s i n - c o n t a i n i n g c h e e s e as an a n t i m i c r o b i a l a g e n t in t h e s e foods.

Table 5 Percentages of nisin-containing cheese (1170 IU of nisin/g) and non-nisin containing cheese (0 IU nisin/g) used to prepare cold-pack cheese spread with the specified concentration of nisin Cheese type 44% Moisture 60% Moisture 0 IU nisin

100 IU nisin

300 IU nisin

0 100

23 77

67 33

0 IU nisin

100 IU nisin

300 IU nisin

0 100

34 66

100 0

% Nisin-containing cheese Non-nisin containing cheese

234

E.A. Zottola et al. / International Journal of Food Microbiology 24 (1994) 227-238

(A)

(B)

1000 -

1000 " • ''l'"

i - i ~ i .C~ •

II

800

~ "''g'"

Control 0 IU

~,'O~ m ,C-, •

300 IU

~

800

• 600 •

~. •

~.,

m.

/

• " ",

.~

;

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600

""...

••

=,

4oo ~ I

%

%°,

• mO

IO01U

?"'"'-.,.

• ;

400

Control 0 IU 1 0 0 IU 3 0 0 IU

%

%

%.

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%0 . . . . . . . . . .

0

200

""'qb") ~%o ~ ..

"'.

%

•

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~

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zo

. . 30

.

Days

. . 40

D-50

o 60

~

, 10

20

30

4'0

"

•,:...,~ _ 50

"m 60

Days

Fig. 1. Recovery of Listeria rnonocytogenes V7 in 44% moisture cold-pack cheese over 56 weeks at: (A) 4°C; (B) 23°C. ( n ) Control, no L. moncocytogenes added; (U) 0 IU, L. monocytogenes added; (o) 100 IU, L. monocytogenes added; (©) 300 IU, L. monocytogenes added.

The cold-pack cheese spreads were inoculated with approximately 1000 test organisms per gram of cheese, this seems to be a more reasonable figure for the organisms used than using much higher numbers, which is not a true picture of what might be encountered in an industrial system. The organisms and the incubation temperatures used for the shelf-stability study as reported here were L. monocytogenes V7 at 4 ° and 23°C, and S. aureus 196E at 23°C, and C. sporogenes PA 3679 at 23°C. The results of the shelf-stability study are shown in Figs. I through 3. An interesting observation on the effect of the nisin-containing cheese on the test microbes was noted. There was an immediate decrease in the numbers. All samples were inoculated with the same number of viable organisms, yet the samples made from the nisin-containing cheese consistently had lower initial numbers than the samples with no nisin-containing cheese. At this time the reasons for this difference are not known, but this immediate effect on these organisms deserves further study. The results shown in Fig. 1 demonstrate the effect that the nisin-containing cheese spreads with 44% moisture had on L. monocytogenes V7 when incubated at 4°C (1A) and 23°C (1B). There was an immediate decrease in numbers as the cheese spreads with nisin had lower initial populations than the cheese spreads

E.A. Zottola et al. / International Journal of Food Microbiology 24 (1994) 227-238

(A) 1oooI~

I

Co.tr,~ 1°°°'(B)l

.11....q ,,,n,. ~ --'(3)-.

'W.. v .... m"'"

0 IU 100 IU 3001u

Ill li

800 "~

~ Control ,,.~,, 0 IU --4-1001u -- ~ • 300 IU

11

"

".

235

BOO

% % %

600 #| I |

400

• |

•

•

% • %11

,

%, •

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i

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oo

"% %

,oo't , ./\" :~ I -

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;|

%| 200

|

(3"

2o0-%

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.:

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•

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10

20

30 Days

40

50

[3---

60

o[

0

'.o"

10

~

20

30 Days

"..... ..... "-'.,,'... 40

50

Fig. 2. Recovery of Listeria monocytogenes V7 in 60 % moisture cold-pack cheese over 56 weeks at: (A) 4°C; (B) 23°C. (£3) Control, no L. moncocytogenes added; ( B ) 0 IU, L. monocytogenes added; (o) 100 IU, L. monocytogenes added; (©) 300 IU, L. monocytogenes added.

without nisin. The reduction in numbers of L. monocytogenes was greater at 23°C than at 4°C. The numbers also decreased in the cheese spreads with no nisin. The decrease that occurred at 23°C could be related to a decrease in pH caused by the growth of the lactic acid bacteria in the cheese spread at the higher temperature. The initial pH of the cold pack cheese spread was adjusted to 5.1 with lactic acid by the end of the sampling time, 56 days, the pH of the spreads at 23°C had decreased to 4.3 whereas the spreads incubated at 4°C, the pH had dropped to only 4.8 to 5.0. At 4°C, a decrease in numbers occurred but the decline stopped at 30 days and the population remained low but at the same level over the remaining 26 days of the time studied. In the higher moisture cheese spreads (Fig. 2) the decline in numbers of L. monocytogenes was greater than that seen in the lower moisture cheese. At 4°C, there was an initial decline followed by a slight increase and another decline until the end of the time studied. At the higher incubation temperature, 23°C, the decline was more rapid and by the 56th day, L. monocytogenes was not recovered from the cheese spread. This decrease in numbers is likely to be attributed to the combined effect of the nisin and the drop in pH of the cheese spread described above.

E.A. Zottola et al. / International Journal o f Food Microbiology 24 (1994) 227-238

236

(A)

(B)

8000

4OOO

' ~ • ""I--

Control

0 iu

m4m

1001U

|

-- , 0 - •

300 [u

I

~ "''Q

I 6000-

3000

I

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Control 0 IU 100 IU 300 IU

I I I

LJ-

• , •2000

4000 •

u ,,

,,,,l

. ,,," i I''

g :" _ , -

2000-

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I

1ooo

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P,." ,I .... !" , • ,. I . .

o

10

.."""~'' 20

,,"

D

10

20

• •

3'0 Days

4~

s'o

oo

30

40

50

60

Days

Fig. 3. Recovery of Clostridium sporogenes PA3679 in cold-pack cheese spread over 56 days. (A) In 44% moisture cold-pack cheese at 23°C; (B) 60% moisture cold-pack cheese spread at 23°C; ([2) Control, no C. sporogenes PA3679 spores added; ( I ) 0 IU, C. sporogenes PA3679 spores added; ( I ) 0 IU, C. sporogenes PA3679 spores added; (e) 100 IU, C. sporogenes PA3679 spores added; (©) 300 IU, C. sporogenes PA3679 spores added.

The effect of the nisin-containing cheese in the cheese spread and the pH change reduced the populations of S. aureus dramatically in the first several days of the study (data not presented). The numbers of S. a u r e u s in the cheese spread containing 44% moisture declined to the point where none were detected after 5 days of storage at 23°C in all of the samples that contained nisin. In the cheese spread without nisin an increase in numbers was noted on the 56th day. In the higher moisture cheese spread, 60% at the same temperature the same rapid decline in numbers was observed. These data suggest that the nisin in the cheese has an immediate effect on the S. a u r e u s cells in the cheese spread, stopping or slowing the growth. The results observed when the cold pack cheese spreads were inoculated with spores of C. s p o r o g e n e s PA 3679 were different than those seen with the other two organisms. The results shown in Fig. 3 indicate that the spores germinated and grew at 23°C in both the high-moisture and low-moisture spreads with the exception of the 60% moisture spread containing 300 IU n i s i n / g where no increase in numbers was noted. Typical spoilage, gas, odor, proteolysis, usually caused by PA 3679 was not noted with the number obtained after 56 days of incubation at 23°C. These results were inconsistent with what had been observed

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with the other organisms and with the same organism in the pasteurized process cheese spread as shown in Table 3. When the pasteurized process cheese spread was made it was given a heat treatment during the process that activated the spores. No such heat treatment was used in the manufacture of the cold-pack cheese spread. To determine if the differences observed in the two cheese spreads were related to the heat process that the pasteurized process cheese spread had received, spores of PA 3679 were given a heat treatment of 80°C for 15 min to activate the spores and then added to cold-pack cheese spread containing 0, 100 and 300 I U nisin/g. The heat activation step resulted in the spores becoming susceptible to the action of the nisin (data not shown). Outgrowth in the nisin-containing cheese was not observed. Thus it becomes apparent that the spores must be activated in some manner before they are sensitive to the action of nisin. One would have to conclude from these results that to assure inactivation of bacterial spores in cold-pack cheese products another preservation process must be used.

4. Conclusions Cheddar cheese m a d e with nisin-producing cultures resulted in a cheese containing approximately 700 IU nisin per g of cheese. The nisin content of the cheese was increased to over 1000 IU nisin per g of cheese by using p H control during growth of the starter culture for cheese manufacture. Utilization of this nisin-containing Cheddar cheese in pasteurized process cheese spread reduced spoilage of the spread by C. s p o r o g e n e s significantly. The data presented suggest that control of L. m o n o c y t o g e n e s and S. a u r e u s in cold pack cheese spreads would be aided by using nisin-containing Cheddar cheese in the formulation. Spore-forming bacteria will not be controlled in cold-pack cheese spreads unless some activation method is used during the manufacture of the spreads. The results of the study presented here suggest that greater utility of bacteriocins and bacteriocin-producing bacteria might be achieved if the bacteriocin was produced 'naturally' in a fermented food. The food product containing the 'naturally occurring' antimicrobial agent could be used as an ingredient in other foods to control unwanted organisms.

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