Increased Nisin in Cheddar-Type Cheese Prepared with pH Control of the Bulk Starter Culture System1

DAIRY FOODS

Increased Nisin In Cheddar-Type Cheese Prepared with pH Control of the Bulk Starter Culture System 1 T. L YEZZI, A. B. AJAO, and E. A. ZOTTOLA Minnesota·South Dakota Dairy Foods Research Center Department of Food Science and Nutrition University of Minnesota S1. Paul 55108 ABSTRACT

Abbreviation key: RSM milk.

Five strains of Lactococcus lactis ssp. lactis and one transconjugant strain of Lactococcus lactis ssp. cremoris, previously shown to be nisin producers, were evaluated for nisin production during growth in pH-controlled media. Four strains showed increased nisin production in pH-controlled 10% reconstituted skim milk. The transconjugant, L. lactis ssp. cremoris IS 102, produced a sixfold increase in nisin, L lactis ssp. lactis NCDO 1402 and CNRZ 148 showed threefold increases, and nisin increased by fourfold in L lactis ssp. lactis CNRZ 150. Lactococcus lactis ssp. lactis NCDO 1404 and ATCC 11454 did not show a discernible increase. All strains had three- to fivefold increases of colony-forming units per milliter when grown in pH-controlled media. Five vats of Cheddar cheese were manufactured using a pH-controlled bulk starter system consisting of strains IS 102 and NCDO 1404. Approximately 20% more nisin was found in cheese prepared with a pHcontrolled bulk starter than in cheese prepared without a pH-controlled bulk starter. The increase in nisin in the cheese could be attributed to higher cell numbers in the pH-controlled bulk starter culture and to carry-over from the inoculum. (Key words: nisin, lactic acid bacteria, cheese)

INTRODUCTION

Received January 19, 1993. Accepted May 18, 1993. 1Published as Paper Number 20,297 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 MinnesotaSouth Dakota Dairy Foods Research Center. 1993 J Dairy Sci 76:2827-2831

= reconstituted skim

Nisin, the inhibitory substance produced by group N streptococci, has utility in food preservation. Many publications 0,4, 5, 6, 10. II, 12, 13, 15, 17) have reviewed and investigated the industrial applications of nisin, including uses in improving food safety and shelf stability of processed cheese. canned foods, alcoholic beverages, meat products, and fresh fish. In the production of cheese, large quantities of lactic acid bacteria are used as starter cultures. Commercially, these bulk starter cultures are produced with or without neutralization of the starter media. As lactic acid-producing bacteria ferment lactose to lactic acid, the buffering capacity of the medium is exceeded, and the pH of the medium begins to drop until the microorganisms are inhibited by the acid produced. More time is required for these starters to repair and to recover from the acidinduced lag phase. If the pH of the media is adjusted above 5.0 during growth, the lag phase can be shortened, and starter culture activity per unit volume can be increased. When a pH-controlled bulk starter system is used, the cheese-making procedure may have to be modified. This modification includes limiting acid development to prevent acid or bitter cheeses, and milling at lower acidities to prevent additional pH drop in the cheese after salting. In a review by Thunell (6), different types of pH control of starter media and advancements in technology are described and discussed. All methods have the net effect of reducing lag time and decreasing the amount of starter needed. Use of nisin-producing strains in a pHcontrolled bulk starter system appears to be a

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YEZZI ET AL.

viable means of increasing the final amount of nisin in a final cheese product. Hurst (8) showed increases in cell biomass and nisin production when nisin-producing strains were grown in pH-controlled media. If the amount of nisin can be increased sufficiently during cheese manufacture, cheese containing a "natural" microbial inhibitor could be used as an ingredient in other food products. The objective of this study was to evaluate the effects of pH-controlled starter media on nisin-producing starter culture systems and to use this technique to modify the starter culture system developed by Roberts (10) and Roberts et al. (II) to increase the amount of nisin in Cheddar-type cheese.

lactis ssp. cremoris were used (Table 1). As needed, strains were thawed and transferred into freshly steamed 10% RSM. Proteinasenegative strains were inoculated into 10% RSM with tryptone. The RSM with tryptone contained .1% tryptone (Difco, Detroit, MI). After transfer, cultures were incubated at 23·C for 16 h. Cultures used to prepare bulk starter were transferred at least twice prior to use to obtain sufficient volume for cheese making. Micrococcus luteus ATeC 10240 was maintained on nisin assay agar slants at 4·C. Transfers onto assay agar slants were incubated at 30·C for 48 h prior to use.

MATERIALS AND METHODS

Individual strains were inoculated into 10% RSM. Samples were grown with and without pH control of the media. The pH was measured using an Orion digital ion analyzer (Orion, Cambridge, MA) equipped with an Orion 91-05 pH electrode. The pH of the bulk cultures was tested and adjusted at hourly intervals to 6.5 ± .2 with 5N NaOH. Samples were removed periodically for nisin analysis and for determination of viable cell counts using M-17 agar (Difco, Surrey, East Molesey, England) supplemented with .5% lactose. Plates were incubated at 30·C for 48 h.

Bacterial Cultures

All strains of nisin-producing lactococci were maintained in frozen storage at the University of Minnesota. Six nisin-producing strains were incubated in 10% reconstituted skim milk (RSM) for 8 to 10 h at 30·C, adjusted to a pH of 6.5 ± .2, frozen, and stored at -50·C. Five nisin-producing strains of Lactococcus lactis ssp. lactis and one nisinproducing transconjugant strain of Lactococcus

Eft.eta of pH-Controlled Media on Nisin-Producing Cultures

TABLE 1. Microorganisms used in this study. Strai'! and source or rererence l

Relevant phenotype2

Culture medium3

PIt- Nip+ Prt+ Nip+ Prt+ Nip+ Prt+ Nip+ PIt- Nip+

RSMT RSM RSM RSM RSMT

Prt+ Nip+ Prt+ Nip-

RSM DVS

Nis'

NAM

lActr.,:occus lactis ssp. lactis NC[IO NCDO CNRZ CNRZ ATCC

1402 1404 148 150 11454

lActococcus lactis ssp. cremoris JSI02 (14) R6044

Micrococcus luteus ATCC 10240

=

=

I ATCC American Type Culture Collection, Rockville, MD; NCDO National Collection of Food Bacteria. Reading. England; CNRZ = Centre National de Recherches Zootechniques, Jouy-en-Josas, France. 2Prt+ Proteinase-positive; PIt- proteinase-negative; Nip+ nisin- producing; Nip- nisin-negative; Nis s nisinsensitive. 3RSMT 10% Reconstituted skim milk (RSM) with .1 % tryptone; DVS direct-vat set; NAM nisin assay medium.

=

=

=

4Chr. Hansen's Lab. Inc.• Milwaukee, WI. Journal of Dairy Science Vol. 76, No. 10. 1993

=

=

=

=

=

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CHEDDAR CHEESE

Bulk St8rter Culture.

Bulk starter cultures were prepared in the following manner. I) An appropriate quantity of 10% RSM was inoculated with a culture incubated at 30·C for 10 h. 2) Bulk cultures were incubated at 30·C for 16 h. 3) The pH of the pH-controlled cultures was adjusted at hourly intervals to 6.5 ± .2 for 16 h at 30·C. 4) Cultures were quickly cooled to 4·C and used within 8 h. Quantification of NI.ln Production

Quantitative estimates of nisin concentrations were determined using the well diffusion assay described by Tramer and Fowler (11). The assay included modifications as set forth by the British Standards Institute (2). The nisin-sensitive indicator strain used was M. luteus ATCC 10240. Samples of milk and whey were prepared by diluting 1 ml of sample into 9 ml of .02N HCI. Cheese and curd samples were prepared as described in the British Standards Institute (2). Samples were analyzed in triplicate. Each well was loaded with 100 ILl of sample and incubated at 30·C for 24 h. After incubation, zones of clearing were measured to the nearest millimeter using a Fisher-Lilly antibiotic Zone Reader (Fisher Scientific, Itasca. IL). Nisin was quantified by comparison of zone diameters with a standard curve. The standard curve was prepared by dilution of the commercially available nisin, Nisaplin$ (Aplin & Bar-

rett Ltd., Trowbridge, London, England), in the appropriate diluent and application to the plates. The standards used were 100, 50, 25, and 2.5 IU/ml. The IoglO IU/ml of each nisin standard was plotted versus the corresponding zone diameter. The relationship was linear over the four standards used. Ch.... Manufacture

Cheddar-type cheese was manufactured from pasteurized whole milk (72°C for 16 s) using 391-kg (860-lb) capacity manually agitated vats (Meyer-Blanke, S1. Louis, MO) in the pilot plant of the Department of Food Science and Nutrition at the University of Minnesota. Five vats of cheese were made using 227 kg (500 Ib) of milk following the procedure described by Kosikowski. (9). The inoculation rate and type are presented in Table 2. The control culture was R604 (Chr. Hansen's Lab, Inc., Milwaukee, WI). When cheese was manufactured, a .5% inoculum of L laetis ssp. eremoris J8I02 and a 1.0% inoculum of L. laetis ssp. laetis NCDO 1404 were used as described by Roberts (10) and Roberts et al. (11). Typically, nisin-producing cultures are slow acid producers. This combination produced acid at rates suitable for Cheddar cheese manufacture. Strain JS 102 was used for its nisin-producing ability and NCDO 1404 for its high acid-producing ability. Batch 2 utilized the starter system described by Roberts (10) and Roberts et at. (11). Batch 3 was similar to batch 2 except that JS l02 was grown in a pH-controlled media. In batch 4,

TABLE 2. Amounts of starter culture with (+) and without (-) pH control used in each of five trials to manufacture Cheddar cheese and final concentration of nisin in the cheese. Strain Al

+ pH Batch

control

Strain B2 - pH control

Strain A - pH control

-

(%)

(IU/g of cheese) SO 334 1170 150 712

X

(no.) 1

2.0

2 3 4 5

Nisin

ControI3

.5 .5 1.0 1.0

1.0 1.0 1.0

a

a

794 868

10 192

ILaetoeoeeus laens ssp. eremoris JS102. 2Laetoeoeeus laetis ssp. lacns NCDO 1404. 3L lactis ssp. eremoris R604. Journal of Dairy Science Vol. 76. No. 10. 1993

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YEZZI ET AL.

only JS 102 grown in pH-controlled media was used. The additional starter activity attained in the pH-controlled bulk starter system was assumed to provide sufficient acid development for Cheddar cheese manufacture. In batch 5, the amount of JSI02 grown in a pH-controlled media was increased to try to increase nisin amounts further in the cheese. Samples were removed at various steps during manufacture for nisin analysis. The number of colonyforming units per milliter of milk or per gram of cheese was determined by transferring samples to pour plates of M-17 agar containing .5% lactose (3). Plates were incubated at 30·C for 48 h, and colonies were counted after incubation. RESULTS AND DISCUSSION

Many L. lactis ssp. lactis strains have been isolated and characterized that are considered to be nisin producers. Researchers (14) have reported that nisin concentrations produced by different strains differed and that certain strains grown in pH-controlled media may produce more nisin (7). The data from this study (Table 3) show differences observed between nisinproducing strains of L. lactis ssp. cremoris and L. lactis ssp. lactis. Strains JSI02, NCOO 1402, CNRZ 148, and CNRZ 150 produced two to five times more nisin when they were

grown in pH-controlled 10% RSM. Strain NCOO 1404 produced more nisin without pH control, and strain ATCC 11454 produced similar amounts of nisin in both situations. The differences between nisin-producing strains JS102 and NCDO 1404 are shown in Figure 1. Strain JSI02 produced more nisin than NCOO 1404 when grown under the same conditions. The effect of pH control of the media is also evident in Figure 1. The amount of nisin increased significantly (approximately sixfold) with J8102 grown in pH-controlled media. When NCDO 1404 was grown in pHcontrolled 10% RSM, less nisin was produced than when the culture was grown without pH control of the media. These data indicate that significant differences exist in nisin production between bacterial strains. When bulk starter cultures prepared by pH control of the starter media were used to make Cheddar-type cheese, amounts of nisin increased. The concentrations of nisin in different lots of cheese are shown in Table 2. Results indicated that approximately 20% more nisin could be produced in Cheddar-type cheese by pH control of the bulk starter culture system developed by Roberts (to) and Roberts

1000

900

800

TABLE 3. Amounts of nisin present in 10% reconstituted skim milk (RSM) after 16 h at 30'C with and without pH control of media. Strain l and source

+ pH

- pH control

control

E

~

- - (IU/ml of milk) - SD SD

X

A. B. C. D. E. F.

JSI02 NCDO NCDO CNRZ CNRZ ATCC

1404 14022 148 150 114542

812 35 714 864 812 484

X

398

137

27

2

147 268 294 220

66

564

277

325 92 398 117

700

34 97 16

.00

500

Z

iii

400

Z

300

200

'00

0 '0

0

lATCC = American Type Culture Collection. Rockville. MD; NCDO = National Collection of Food Bacteria, Reading. England; CNRZ = Centre National de Recherches Zootechniques, Jouy-en-Josas. France. 2RSMT = 10% Reconstituted skim milk with .1 % tryptone. Journal of Dairy Science Vol. 76. No. 10. 1993

TIME

12

(h)

Figure 1. Nisin production of Lactococcus lactis ssp. cremoris JS102 in 10% reconstituted skim milk (RSM) without (0) and with pH control of medium (II). Nisin production of Lactococcus lactis ssp. lactis NCDO 1404 in 10'Jb RSM without (e) and with pH control of medium (0).

CHEDDAR CHEESE

et al. (11). When the whey was drained from the curd, the nisin content of the whey was approximately 100 ill of nisin/ml of whey. The whey was discarded, and no further analysis was perfonned. CONCLUSIONS

Data from this study suggest that bulk starter cultures prepared in pH-controlled media and subsequently used to manufacture cheese can result in higher concentrations of nisin in the cheese. Different strains of nisinproducing lActococcus ssp. produced different quantities of nisin under the same conditions. Nisin concentrations are strain-dependent. The amount of nisin produced by strains IS 102, NCDO 1402, CNRZ 148, and CNRZ 150 could be increased by growth under pH control. Strains NCDO 1404 and ATCC 11454 did not show appreciable increase in nisin production using pH-controlled media. The pHcontrolled bulk starter cultures could be used to increase the amount of nisin in Cheddar cheese. However, each individual strain must be tested to detennine whether nisin has increased in pH-controlled media. REFERENCES

1 Berridge, N. J. 1953. The antibiotic nisin and its use in the maldng and processing of cheese. Chern. Ind. 1953:1158. 2 British Standards Institute. 1974. Methods for the Estimation and Differentiation of Nisin in Processed Cheese. Publ. 4020, Br. Standards Inst., London, Engl. 3 Busta, F. F., E. H. Peterson, D. M. Adams, and M. G. Johnson. 1984. Colony count methods. Page 66 in

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Compendium of Methods for the Microbiological Examination of Foods. M. L. Speck, ed. Am. Public Health Assoc. Washington, DC. 4 Calderon, C., D. L. Collins-Thompson, and W. R. Usborne. 1985. Shelf-life of vacuum-packaged bacon treated with nisin. J. Food Prot. 48:330. 5 Chung, K. T., J. S. Dickson, and J. D. Crouse. 1989. Effect of nisin on growth of bacteria attached to meat. Appl. Environ. Microbiol. 55:1329. 6 Fowler, G. G. 1979. The potential of nisin. Food Manur. 54:57. 7 Hirsch, A. 1951. Growth and nisin production of a strain of Streptococcus lactis. 1. Gen. Microbiol. 5: 208. 8 Hurst, A. 1981. Nisin. Adv. Appl. Microbiol. 27:85. 9 Kosikowski, F. V. 1982. Cheese and Fermented Milk Foods. F. V. Kosikowski and Assoc., Brooktondale. NY. 10 Roberts, R. F. 1991. Development of a nisinproducing starter culture for use during Cheddar cheese manufacture to inhibit spoilage in high moisture pasteurized process cheese spreads. Ph.D. Diss.• Univ. Minnesota, St. Paul. 11 Roberts, R. F., E. A. Zottola, and L. L. McKay. 1992. Use of a nisin-producing starter culture suitable for Cheddar cheese manufacture. J. Dairy Sci. 75:2353. 12 Scott, V. N., and S. L. Taylor. 1981. Effect of nisin on the outgrowth of Clostridium botulinum spores. J. Food Sci. 46:117. 13 Somers, E. B., and S. L. Taylor. 1987. Antibotulinal effectiveness of nisin in pasteurized process cheese spreads. J. Food Sci. 46:121. 14 Steele, J. L., and L. L. McKay. 1986. Partial characterization of the genetic basis for sucrose metabolism and nisin production in Streptococcus lactis. Appl. Environ. Microbiol. 51:57. 15 Tanaka, N., E. Traisman, P. Plantilnga, L. Finn, W. Flom, L. Meske, and J. Guggisberg. 1986. Evaluation of factors involved in antibotulinal properties of pasteurized process cheese spreads. 1. Food Prot. 49:526. 16 Thunell, R. K. 1988. pH-Controlled starter: a decade reviewed. Cult. Dairy Prod. J. 23:(3)10. 17 Tramer, J., and G. G. Fowler. 1964. Estimation of nisin in foods. 1. Sci. Food Agric. 15:522.

Journal of Dairy Science Vol. 76, No. 10. 1993