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Department of Animal Science and Food and Agricultural Products Center Oklahoma State University, Stillwater 74078-6055
Corresponding author: S. E. Gilliland; e-mail: seg{at}okstate.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Lactobacillus delbrueckii ssp. lactis • cottage cheese • psychrotrophic spoilage
Abbreviation key: CVT = crystal violet tetrazolium chloride, LBS = lactobacillus selection, MRS = de Man, Rogosa, Sharpe.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability of lactic acid bacteria to preserve food and prevent the growth of undesirable organisms has been studied for several years. Many lactic acid bacteria have the ability to produce substances that are inhibitory at low temperatures to the growth of other bacteria (including pathogens and spoilage organisms). An important factor for these organisms in food systems is their ability to produce inhibitory substances at refrigeration temperatures while not growing (Daly et al., 1972; Gilliland and Speck, 1975; Juffs and Babel, 1975; Martin and Gilliland, 1980). Various researchers have reported the use of Lactobacillus spp. as one of the cultures effective at inhibiting the growth of psychrotrophic and pathogenic organisms at refrigeration temperatures. Gilliland and Ewell (1983) reported that Lactobacillus lactis was inhibitory to psychrotrophic organisms in raw milk at 5°C. Brashears et al. (1998) showed that Lactobacillus delbrueckii ssp. lactis was effective at reducing numbers of Escherichia coli O157:H7 on raw chicken stored at 5°C. The compound produced by L. delbrueckii ssp. lactis responsible for this was identified as hydrogen peroxide. Premi and Bottazzi (1972) found that L. lactis produced more hydrogen peroxide than other species of lactobacilli. Yap and Gilliland (2000) reported that L. delbrueckii ssp. lactis RM2-5 produced more hydrogen peroxide than did other strains of L. delbrueckii ssp. lactis tested. The ability of this organism to produce more hydrogen peroxide at refrigeration temperatures while not growing allows for its potential use as a biopreservative in refrigerated foods.
The objective of this study was to determine if cells of L. delbrueckii ssp. lactis RM2-5 would be effective at inhibiting the growth of spoilage organisms in cottage cheese at 7°C, thus extending the product’s shelf life.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Enumeration of Bacteria
The lactobacilli were enumerated using lactobacillus selection (LBS) agar prepared from individual ingredients according to the manufacturer’s specifications (BBL, Cockeysville, MD). Plates were prepared using the pour-plate technique with an overlay, placed in plastic bags, flushed with carbon dioxide, sealed, and incubated at 37°C for 48 h.
Crystal violet tetrazolium chloride (CVT) agar, which is selective for gram-negative microorganisms, was used for the enumeration of P. fluorescens (Swanson et al., 1992). It was prepared by adding 1 mL of a 0.1% ethanolic solution of crystal violet to 1 L of plate count agar (Difco Laboratories). To better see the colonies in the agar, 1 mL/100 mL of 0.5% aqueous solution of 2,3,5 triphenol tetrazolium chloride was added to the molten agar before plating. The plates were incubated aerobically at 15°C for 5 d.
Populations of lactobacilli in frozen concentrated cell suspension were determined by plating on MRS agar with an overlay of the same medium (MRS broth supplemented with 1.5% agar). Plates were incubated aerobically for 48 h at 37°C.
Sample preparation and dilutions for plating were done using procedures from the Compendium of Methods for the Microbiological Examination of Foods (Swanson et al., 1992). Dilutions were made using sterile peptone diluent containing 0.1% peptone and 0.001% antifoam A (Sigma-Aldrich, St. Louis, MO) in distilled water. Initial dilutions were made using a Dilumat 3 automatic dilution dispenser (AES Laboratoire, Cambourg, France). For the initial dilution, approximately 11 g of cottage cheese was weighed into a stomacher bag on the Dilumat 3, which then automatically added enough sterile diluent to create a 1:10 dilution. The sample was then pummeled for 60 s in a masticator (RUL Instruments, Barcelona, Spain). The remaining dilutions were done using 99 mL dilution blanks. After the appropriate incubation period, colonies on plates were enumerated using a Quebec Darkfield colony counter (Darkfield-model 3325, Buffalo, NY).
Preparation of Cultures for Treatments
The RM2-5 strain used in the initial experiments was grown in MRS broth for 18 h at 37°C under static conditions, harvested by centrifugation at 5000 x g for 30 min at 2 to 3°C, and resuspended in 15 mL of cold, sterile 10% nonfat milk solids. The resulting concentrated culture was held on ice until used (not more than 30 min).
Cultures of L. delbrueckii ssp. lactis RM2-5 used in all subsequent experiments were prepared as frozen concentrated suspensions. Thirty milliliters of a freshly prepared culture of lactobacilli were inoculated into 3000 mL of MRS broth in a Bioflo 2000 fermentor connected to a pH 2000 control module (New Brunswick Scientific Co., Edison, NJ) and incubated at 37°C for 6 h. The pH was not permitted to fall below 5.00 by automatic addition of a solution of 20% sodium carbonate in 20% ammonium hydroxide as described by Brashears and Gilliland (1995). After incubation, the cells were harvested by centrifugation at 5000 x g for 30 min at 2 to 3°C and resuspended in enough cold, sterile 10% nonfat milk solids to yield a population of approximately 1.0 x 1010 cfu/g. Sterile glass beads (2 mm diameter) were used to aid in the resuspension of the pellet into the milk. The resulting concentrated suspension was aseptically dispensed in 2-g aliquots into sterile cryogenic vials and frozen in liquid nitrogen at –196°C until used. After 1 d of storage in liquid nitrogen, 1 vial was removed, thawed (by submersion in 1 L of tap water at room temperature for 10 min), and plated on MRS agar to determine the population of the L. delbrueckii ssp. lactis RM2-5. Before opening each vial, the exterior was sanitized with 70% alcohol. For use in the experiments, the appropriate number of vials needed were thawed then held on ice (less than 1 h) until used.
The P. fluorescens was a freshly prepared culture that had been subcultured 3 times in tryptic soy broth before the experiment. On the day of the experiment, the culture was held on ice until used in the treatments (less than 30 min). The inoculum used in the treatments was made by preparing a 1:1000 dilution of P. fluorescens and adding the necessary amount to the cottage cheese cream to yield a population of approximately 1.0 x 102 cfu/g in the cheese.
Preparation of Treatments for Storage
Dry cottage cheese curd was obtained from a commercial dairy processing plant on the day of manufacture and used the same day in experiments. Half-and-half (10.5% fat) was used as the creaming mixture, and was purchased from a local supermarket on the morning each experiment began, and held in the refrigerator until use. Immediately before use, it was repasteurized by transferring it to Pyrex bottles (Fisher Scientific, Pittsburgh, PA), which were placed in boiling water for 30 min, and then rapidly cooled in ice water.
Initial experiment using nonfrozen RM2-5.
In the initial experiments, 5 treatments were prepared for storage. The dry curd cottage cheese was mixed and dispensed with a sterile spatula in 300-g portions in each of 5 sterile 1000-mL beakers labeled 1 to 5. To inoculate cream, 3 mL of a 1:1000 dilution of P. fluorescens was added to 960 mL of freshly pasteurized half-and-half in a sterile flask, mixed, and then dispensed in 175-g amounts into each of 5 sterile flasks. Sufficient amounts of the cell suspension of L. delbrueckii ssp. lactis RM2-5 were added to yield populations of 0, 5.0 x 107, 1.0 x 108, 5.0 x 108, and 1.0 x 109 cfu/g in the 5 flasks (treatments 1 to 5, respectively). The contents of flasks 1 to 5 were added to the dry curd in beakers 1 to 5 respectively. The cottage cheese mixture in each beaker was mixed and dispensed, using a sterile spatula, in approximately 100-g portions into 4 appropriately labeled sterile 150-mL beakers (i.e., 4 beakers for each of 5 treatments). All beakers were stored at 7°C. One beaker of each treatment was removed on d 0, 7, 14, and 21 for enumeration of bacteria and for pH measurement. Two replicates were done for this experiment.
Effect of RM2-5 from frozen concentrate.
All subsequent experiments were done using the frozen concentrated cell suspensions of L. delbrueckii ssp. lactis RM2-5. Samples were prepared as described in the previous section except that populations of RM2-5 were 0, 5.0 x 107, 1.0 x 108, 2.0 x 108, and 5.0 x 108 cfu/g.
Effect of RM2-5 on uninoculated cottage cheese.
Two separate trials were conducted in which no spoilage organisms were inoculated into the cottage cheese. In the first experiment, dry curd was mixed and 300-g portions were dispensed into each of 5 beakers. Cream (half-and-half) was pasteurized as described previously and dispensed in 175-g amounts into 5 sterile flasks. The appropriate amounts of frozen concentrated cell suspension of RM2-5 were added to yield populations of 0, 5.0 x 107, 1.0 x 108, 2.5 x 108, and 5.0 x 108 cfu/g in the cheese. Cream was added to appropriately labeled beakers containing dry curd, mixed, and aseptically dispensed in approximately 50-g amounts into each of 5 appropriately labeled, sterile 150-mL beakers. All beakers were stored at 7°C. One beaker of each treatment was removed on d 0, 7, 14, 21, and 28 for enumeration of bacteria and measurement of pH. The experiment was replicated 3 times.
In the second experiment, samples were prepared in the same way as for the first experiment except only 3 treatments (0, 5.0 x 107, and 1.0 x 108 cfu/g of lactobacilli) were prepared and 8 portions (50 g each) of each treatment were dispensed for storage to permit analysis on d 0, 7, 14, 21, 28, 35, 42, and 49. Three replicates were done.
pH Analysis of Samples
The pH of each sample was measured on each sample day. After removing 11 g for plating, the remaining portion was used to determine pH using an AR 25 dual channel pH/ion meter (Fisher Scientific).
Statistical Analyses
Statistical analysis was conducted for all experiments as a split plot in a randomized block design using PROC MIXED and LSMEANS, where replicates were blocks, treatments served as the main units, and storage time was the subunit. This analysis was conducted using SAS software (SAS Institute, 1985). Least significant differences were used to compare means at the 5% confidence level. Factorial arrangement of the analysis depended on the experiment, because the number of treatments, storage times, and number of replicates varied among experiments.
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RESULTS |
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). As the amount of lactobacilli added increased, the amount of spoilage organisms present (as indicated by plate counts on CVT agar) significantly declined during storage (P < 0.05). On d 0, there was no significant difference (P > 0.05) among the 5 treatments although there was a tendency for lower numbers of spoilage organisms in the samples containing the highest numbers of added lactobacilli. However, by d 7, spoilage organisms in the control (no lactobacilli) had increased more than 5 log cycles, whereas treatments 2 and 3, which contained the 2 smallest amounts of lactobacilli, had only increased 3 log cycles. Treatments 4 and 5, which contained the largest amounts of lactobacilli, did not exhibit any increase in numbers of spoilage bacteria by d 7. By d 14, the first 3 treatments, which contained no or low levels of lactobacilli, had CVT counts of approximately 9.7 log10 cfu/g. Treatments 4 and 5 had significantly lower (P < 0.05) CVT counts on d 21 than did treatments 1, 2, and 3. Treatment 4 had a count of 6.3 log10 cfu/g on d 21; treatment 5 had a count of <1.00 log10 cfu/g. The results indicate that, if present in adequate numbers, lactobacilli inhibit growth of P. fluorescens in cottage cheese. Lactobacilli enumerated on LBS agar remained moderately constant with only slight decreases over time (data not shown). The pH did not change over time (data not shown).
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Cells from the frozen concentrated suspension, however, did not appear to be as effective as the nonfrozen cells (Table 2) in controlling growth of P. fluorescens in cheese. However, there were significant differences among treatments over the 21-d storage time. On d 0, there was no significant difference (P > 0.05) among treatments, but by d 7 there was a 4 log cycle difference between the control and treatment 5 [which contained the highest number (5.0 x 108 cfu/g) of lactobacilli]. By d 21, although counts of spoilage bacteria were high in all samples, there were significantly fewer (P < 0.05) in treatment 5 than in the control. The counts of lactobacilli and pH remained constant over the 21-d storage period (data not shown).
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). As the number of cells of lactobacilli increased, there were significant (P < 0.05) decreases in the growth of spoilage organisms over the 28-d period. In this trial, no spoilage organisms were detected in treatment 5 (5.0 x 108 cfu/g) until d 14, whereas the control had a count of 4.5 log10 cfu/g by d 7, indicating the effects of RM2-5 on the growth of gram-negative bacteria, which can cause spoilage of cottage cheese. On d 28, there was a 3 to 4 log cycle difference between the control and the 3 treatments with the 3 highest levels of RM2-5. The sporadic growth of molds in the samples was responsible for some of the counts observed on CVT agar. This suggests that the lactobacilli were not effective in controlling growth of molds. In this experiment, the numbers of lactobacilli and pH changed very little during storage.
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). As in the previous trial, mold growth became a factor in some samples. However, as in the other experiments, it was apparent that lactobacilli were effective at slowing the growth of spoilage organisms over an extended period. In treatment 3 (1 x 108/g), after a slight peak of spoilage organisms on d 21, the population remained constant at approximately 2 log10 cfu/g over the rest of the storage period with slight numerical (not significant) decreases. The counts of spoilage bacteria on CVT agar for control and treatment 2 increased significantly (P < 0.05) over time. After 49 d, the pH values showed variation among treatments not seen in previous experiments. On d 0, all treatments had a pH of ~5.00; after 49 d, treatment 1 had a pH value of 5.7, treatment 2 had a value of 5.5, and treatment 3 remained moderately constant at 5.2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The potential for the beneficial effects of adding L. delbrueckii ssp. lactis RM2-5 to creamed cottage cheese was demonstrated in this study. The results indicate significant (P < 0.05) inhibition of growth of spoilage organisms in cottage cheese during storage at 7°C. However, relatively high numbers of the lactobacilli were required. Based on results from earlier studies, strain RM2-5 produces considerable hydrogen peroxide at refrigeration temperatures (Yap and Gilliland, 2000). Because only lactobacilli were added to the cheese, it is probable that hydrogen peroxide produced by RM2-5 was responsible for the inhibition of spoilage organisms over the course of this study. Throughout these experiments, varying amounts of RM2-5 were added in different experiments. The purpose of adding different amounts was to find the least amount of lacto-bacilli necessary to provide effective results. Weber and Broich (1986) reported that the more beneficial bacteria added the better chance one has in controlling the bacteria that cause spoilage. Because relatively high numbers were required to achieve the greatest degree of inhibition, more research is needed to enhance the antagonistic effect so that fewer numbers of RM2-5 lacto-bacilli are needed to achieve complete inhibition for a long period.
Use of a combination of antimicrobials might be effective. Several researchers have reported that naturally occurring antimicrobials produced by certain bacteria are effective at controlling undesirable microorganisms in cottage cheese (Weber and Broich, 1986; Tortorello et al., 1991). In a study using naturally occurring substances produced by bacteria and fungi, Tortorello et al. (1991) were able to effectively extend the shelf life of cottage cheese 2 wk beyond its code date. One antimicrobial substance naturally produced by Propionibacterium shermanii is Microgard (skim milk fermented by P. shermanii then dried), a commercially available product that is often used in cottage cheese production (Daeschel, 1989). Weber and Broich (1986) showed the effectiveness of Microgard in controlling gram-negative spoilage organisms in cottage cheese. They also reported fewer instances of mold in cottage cheese that contained Microgard. Another antimicrobial currently added to cottage cheese is potassium sorbate. Much research has been done to show the effectiveness of potassium sorbate in controlling molds and some bacteria in cottage cheese. Because Microgard and potassium sorbate have no effect on the growth of gram-positive bacteria (Daeschel, 1989), it would be possible to develop a combination of Microgard, potassium sorbate, and L. delbrueckii ssp. lactis RM2-5, which would have the potential to control a variety of spoilage organisms, including gram-negative bacteria and molds, for an extended period.
The results from this study indicated that, when used alone and at an appropriate level, L. delbrueckii ssp. lactis RM2-5 was very effective at controlling growth of spoilage organisms in refrigerated cottage cheese. Although most treatments eventually showed evidence of spoilage (as evidenced by slime formation or surface mold growth) by the end of the storage period, it was still evident that in the treatments containing RM2-5, it usually took longer for the cottage cheese to exhibit spoilage defects. In most experiments on the final day of storage, the samples containing higher numbers of the lactobacilli had CVT counts several log cycles lower than controls. Results also indicated that pH values for most experiments remained moderately constant over time. These results indicate the effectiveness of using L. delbrueckii ssp. lactis RM2-5 in creamed cottage cheese to control spoilage organisms and the potential for its use in combination with other antimicrobial products to extend shelf life. Although no sensory evaluations were included in this study, it is not likely that lactobacilli would cause any adverse flavors in the cheese. In related experiments using this organism, no off-flavors were noted.
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FOOTNOTES |
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Current address: Bar-S Foods, Clinton, OK 73601-4148.
Received for publication August 3, 2004. Accepted for publication December 11, 2004.
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REFERENCES |
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