Shelf-Life Evaluation of Natural Antimicrobials for Concord and Niagara Grape Juices

Transcription

1 72 Journal of Food Protection, Vol. 76, No. 1, 2013, Pages doi: / x.jfp Copyright G, International Association for Food Protection Shelf-Life Evaluation of Natural Antimicrobials for Concord and Niagara Grape Juices P. SIRICURURATANA, M. M. IYER, D. C. MANNS, J. J. CHUREY, R. W. WOROBO, AND O. I. PADILLA-ZAKOUR* Department of Food Science, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456, USA MS : Received 27 March 2012/Accepted 14 September 2012 ABSTRACT This study was conducted to evaluate the effectiveness of natural antimicrobials for shelf-life extension of cold-filled still and carbonated Concord and Niagara grape juices, which have traditionally been preserved with chemical preservatives. Commercial juices were inoculated with a spoilage yeast cocktail of Dekkera, Kluveromyces, Brettanomyces, and Zygosaccharomyces at 10 2 and 10 4 CFU/ml. The following agents were added to still juices: no preservative (negative control), 0.05% potassium sorbate plus 0.05% sodium benzoate (positive control), 0.1 or 0.2% cultured dextrose, 250 ppm of dimethyldicarbonate (DMDC), 10 or 20 ppm of natamycin, and 250 ppm of DMDC plus 5 or 10 ppm of natamycin. Carbonated juice was treated with the negative control, positive control, and 250 ppm of DMDC plus 10 ppm of natamycin. Microbial stability of samples was assessed every 2 weeks during 6 months of storage at 21uC by yeast enumeration and measurement of turbidity, ph, and ubrix. Juices were deemed spoiled when yeast counts exceeded 10 6 CFU/ml. Cultured dextrose was not effective at levels tested in both types of juice. The most promising results were obtained with DMDC and natamycin combination treatments in still Niagara juice and in carbonated Concord and Niagara juices. In these treatments, shelf-life extension similar to that of the positive control (153 to 161 days) was achieved while maintaining similar turbidity, ph, and ubrix. Spoiled juices had lower ph and ubrix values and higher turbidity due to microbial activity and increased in microbial levels. Because of increased consumer preferences for allnatural foods and beverages, natural antimicrobial systems for preservation of Concord and Niagara grape juices were investigated. Modern consumer predilections are increasingly demanding that foods be of high quality, free from chemical preservatives, and mildly processed, possess a lengthy shelf life, be palatable, and comply with food legislation (3). The application of preservative treatments for grape juice is particularly useful in production of coldfilled juices. Hot-filled juices are bottled at 70 to 80uC for improved protection against spoilage, but this final heat treatment is not applied to cold-filled juices, resulting in a much greater need for preservatives (2). Wild and preservative-resistant yeasts (e.g., Brettanomyces, Schizosaccharomyces, and Zygosaccharomyces) present major microbial spoilage problems for cold-packed and carbonated grape juice (12). Growth of these spoilage yeasts often results in alcohol production and off-flavors. Gas production from microbial activities can cause glass bottles to explode due to the high internal pressures, potentially presenting a consumer hazard. Various compounds are used to extend the shelf life of fruit products, and many are used in combination. These antimicrobials can be classified as traditional or naturally occurring (5). Traditional preservative systems that rely on sorbate and benzoate are ineffective * Author for correspondence. Tel: ; Fax: ; oip1@cornell.edu. for preventing the growth of Zygosaccharomyces (20, 21). Sorbate and benzoate are frequently used together for their synergistic effect as a preservative system for juices and other beverages. However, growing consumer concerns about chemical preservatives because of their potential for toxicity and allergic reactions has resulted in decreased consumer acceptance of these compounds. Dimethyldicarbonate (DMDC), an approved food additive in 100% juice products (17) when added during bottling, is a potent and broad-acting antimicrobial compound that quickly degrades in water matrices. After exercising its antimicrobial effect, DMDC is hydrolyzed to carbon dioxide and trace amounts of methanol at levels not considered toxicologically significant. These by-products yield no residual odors or flavors and have not caused allergic responses (9). Because of the production of methanol, legal limits on the use of DMDC have been set at 250 ppm in fruit juices (18). The effective antimicrobial agent is the parent compound, DMDC, and therefore the primary protective effect occurs during the initial dosing before DMDC degradation (7). As a result, DMDC does not provide longterm protection against recontamination or later outgrowth of surviving microorganisms. DMDC is particularly active when the ph is low and/or when the concentration of DMDC or the product temperature is high (10). DMDC has been used successfully to specifically inhibit the spoilage of grape juice. Terrell et al. (14) investigated the impact of various concentrations of DMDC, sulfur dioxide (SO 2 ), and sorbic

2 J. Food Prot., Vol. 76, No. 1 NATURAL ANTIMICROBIALS FOR GRAPE JUICE 73 acid, separately and in combination, on the growth of Saccharomyces cerevisiae strain Montrachet in Venus grape juice. These authors investigated initial inoculum levels of 2, 200, and 20,000 CFU/ml in grape juice stored at both 21 and 31uC. Fermentation was inhibited for more than 1 year when 0.8 mm DMDC (,108 ppm) was applied to the 2 and 200 CFU/ml treatments at 21uC and to all inoculum levels at 31uC (8, 12). TheefficacyofDMDCimprovedwithincreasing temperatures, in contrast to the action of SO 2 and sorbic acid, which have reduced antimicrobial activity at higher temperatures. DMDC is a beverage sterilant and a processing aid and does not require specific labeling on the finished product. Natamycin, an antimicrobial polyene macrolide produced from the controlled fermentation of dextrose by Streptomyces natalensis, has been used as a natural preservative in certain foods and beverages to prevent spoilage from growth of yeasts or molds but not from growth of bacteria. Given its low solubility in water (40 mg/ml), natamycin is more commonly used as a surface treatment but has been reported as an effective preservative in both pasteurized and unpasteurized juices, most likely because of the very low MIC required for effectiveness against most mold species (6, 14). Natamycin does not affect the flavor of fruit juices (6) when applied at the suggested dose of 2.5 to 10 ppm (14). Thomas and Delves-Broughton (15) observed that 10 to 40 ppm delayed the growth of most of the yeast strains studied by more than 63 days in apple, orange, and pineapple juices. Natamycin is available commercially under the trade names Natamax (Danisco A/S Corp., Copenhagen, Denmark) and Delvocid (DSM Food Specialties USA, Charlotte, NC), both of which contain approximately 50% natamycin blended with lactose. MicroGard 200 (Danisco A/S Corp.) inhibits yeasts and bacteria. It is used in the food industry and has regulatory approval for beverages as a yeast inhibitor. MicroGard 200 is produced by the fermentation of dextrose by Propionibacterium shermanii or specific lactococci to produce antimicrobial compounds such as diacetyl, lactic acid, propionic acid, acetic acid, and other unidentified compounds in the range of 700 Da (1, 19). Because of growing concerns about chemical preservatives, natural antimicrobials could be used instead of conventional preservatives to satisfy consumer demands. The effectiveness of three alternative antimicrobials, DMDC, cultured dextrose, and natamycin, both alone and in combination, was tested in comparison to that of the traditional chemical preservatives benzoate and sorbate in Concord and Niagara grape juices to assess their effect on microbial stability and product quality. MATERIALS AND METHODS Juice and preservatives. Single-strength not-from-concentrate commercial Concord and Niagara grape juices were purchased from local stores (Wegmans, Geneva, NY) and used for all studies. Two grape juice products, still and carbonated (three volumes of CO 2 ), were used as a test system for various antimicrobials. Potassium sorbate and sodium benzoate (Fisher Scientific, Pittsburgh, PA) were used together as traditional preservatives. Three natural preservatives were tested: (i) cultured dextrose in the form of MicroGard 200, (ii) natamycin in the form of Natamax, and (iii) DMDC in the form of Velcorin (Bayer Corp., Pittsburgh, PA). Juice preparation. Juices were inoculated with a cocktail of Zygosaccharomyces, Kluveromyces, Dekkera, and Brettanomyces (juice and beverage isolates from the culture collection of R. Worobo). The yeasts were grown for 18 h at 30uC in potato dextrose broth (Difco, BD, Sparks, MD). The inoculum was diluted in sterile peptone water at either 100 or 10,000 CFU/ml, representing low and high contamination levels. The preservatives were then added, and the juices were packed into 50-ml presterilized polypropylene tubes and stored at 21uC for 24 weeks. Only DMDC was added into prepacked juices immediately before being capped. Potassium sorbate and sodium benzoate, each at a concentration of 0.05% (wt/vol), were used together as a positive control in both still and carbonated juices; the negative control contained no preservatives. In still juice, 0.1 and 0.2% (wt/vol) cultured dextrose, 250 ppm of DMDC, and 10 and 20 ppm of natamycin were tested. A combination of 250 ppm of DMDC with 5 or 10 ppm of natamycin was tested in still juice, and a combination of 250 ppm of DMDC with 10 ppm of natamycin was tested in carbonated juice. Quality evaluation. Samples were tested every 2 weeks for ph, ubrix, turbidity, and microbial counts. The ph was measured with an Orion 3 Star Series benchtop model ph meter (Thermo Electron Corp., Beverly, MA), and ubrix was measured with an Auto ABBE refractometer (Leica Inc., Buffalo, NY). Turbidity was measured with a 2100P turbidimeter (Hach Co., Loveland, CO) and reported in nephelometric turbidity units (NTU). Carbonated samples were sonicated in a Branson 2200 sonicator (Fisher Scientific, Agawam, MA) to remove the air bubbles before turbidity was measured. Free SO 2 in both starting juice matrices was measured immediately upon bottle opening with the FIAstar 5000 (FOSS Analytical A/S, Eden Prairie, MN) using the method described in application note 5270 (13). Total yeasts were enumerated in duplicate after serial dilution on 3.9% potato dextrose agar acidified to ph 3.5 with 10% tartaric acid. All plates were incubated at 30uC for 72 h before enumeration. An upper limit of 10 6 CFU/ml was used as an indicator for spoilage. Means of all duplicate measurements were calculated for each juice at each storage time. Individually comparing each treatment against its respective control, a standard two-tailed Student t test was used to evaluate the changes in shelf life provided by the various antimicrobial additions. RESULTS AND DISCUSSION Quality evaluation. The Niagara grape juice used in this study had ppm of free SO 2, whereas the Concord grape juice contained only ppm. This difference is most likely due to the addition of sulfites to the Niagara grapes during mechanical harvesting to inhibit enzymatic browning. Initial ph, ubrix, and turbidity values of each grape juice are shown in Tables 1 and 2. Concord juice had higher ph, ubrix, and turbidity values than did Niagara juices. Spoiled juices had decreased ph (3.07 to 3.12 for Niagara and 3.20 to 3.33 for Concord) and ubrix (6.64 to for Niagara 6.20 to ubrix for Concord) but increased turbidity (15 to 890 NTU for Niagara and 80 to more than 1,000 NTU for Concord). These changes were due to the metabolic activity of the increasing populations of yeasts.

3 74 SIRICURURATANA ET AL. J. Food Prot., Vol. 76, No. 1 TABLE 1. Values for ph, ubrix, and turbidity in cold-filled grape juices treated with various antimicrobials on day 0 Juice Treatment ph ubrix Turbidity (NTU) Concord Low yeast inoculum Control % Sorbate z 0.05% benzoate ppm of DMDC a % Cultured dextrose % Cultured dextrose ppm of natamycin ppm of natamycin High yeast inoculum Control % Sorbate z 0.05% benzoate NA b 250 ppm of DMDC NA 0.1% Cultured dextrose NA 0.2% Cultured dextrose NA 10 ppm of natamycin NA 20 ppm of natamycin NA Niagara Low yeast inoculum Control % Sorbate z 0.05% benzoate ppm of DMDC % Cultured dextrose % Cultured dextrose ppm of natamycin ppm of natamycin High yeast inoculum Control % Sorbate z 0.05% benzoate ppm of DMDC % Cultured dextrose % Cultured dextrose ppm of natamycin ppm of natamycin a DMDC, dimethyldicarbonate. b NA, not available. Upon addition to the juice matrix, the cultured dextrose material significantly increased the initial turbidity of both juices compared with the control and other test matrices (Table 1), indicating that this treatment might not be suitable for clear beverages. However, perhaps as a result of its low level of solubility (Fig. 1) the cultured dextrose supplement had the lowest capacity for juice preservation; addition of up to 0.2% (wt/vol) MicroGard 200 did not effectively extend the shelf life beyond that of the negative control (no preservatives) in either the Concord or Niagara grape juices. For still Concord grape juice, none of the antimicrobials at the concentrations tested could prolong the shelf life to the same degree as did the traditional preservatives (sorbate and benzoate) in the positive control (Fig. 1A and 1B). However, the two assessed concentrations of natamycin TABLE 2. Values for ph, ubrix, and turbidity in cold-filled Niagara grape juices treated with antimicrobial systems on day 0 Niagara juice Treatment ph ubrix Turbidity (NTU) Still, low yeast inoculum Control % Sorbate z 0.05% benzoate ppm of DMDC a z 10 ppm of natamycin ppm of DMDC z 5 ppm of natamycin Still, high yeast inoculum Control % Sorbate z 0.05% benzoate ppm of DMDC z 10 ppm of natamycin ppm of DMDC z 5 ppm of natamycin Carbonated, high yeast inoculum Control % Sorbate z 0.05% benzoate ppm of DMDC a DMDC, dimethyldicarbonate.

4 J. Food Prot., Vol. 76, No. 1 NATURAL ANTIMICROBIALS FOR GRAPE JUICE 75 FIGURE 1. Microbial stability of Concord and Niagara juices inoculated with low and high yeast levels and treated with various antimicrobials. (A) Concord juice with low yeast inoculum; (B) Concord juice with high yeast inoculum; (C) Niagara juice with low yeast inoculum; (D) Niagara juice with high yeast inoculum. helped delay yeast growth (a ~ 0.05); the microbial count increase began later than that in the negative control samples. DMDC was less efficacious (a ~ 0.1); it delayed yeast spoilage for an additional 6 days beyond that in the negative control samples. Shelf-life extension was slightly improved when a combination treatment of DMDC plus natamycin was used in the low yeast inoculum treatment (Fig. 2A) compared with each of the individual treatments (a ~ 0.05). This effect was strengthened when DMDC was combined with increasing concentrations of natamycin. In low yeast inoculated Concord juice treated with a combination of DMDC and natamycin, yeasts were not detectable early in the storage period (up to 49 days) and then began to increase. Thus finding suggests that the inoculated yeasts were initially inhibited by these antimicrobials but recovered and resumed growth during storage when the antimicrobial effect disappeared. When the concentration of DMDC was held constant, an increasing concentration of natamycin increased the shelf life. After treatment with high yeast inoculum, the combination of DMDC and a lower concentration of natamycin (5 ppm) delayed Concord juice spoilage, but the shelf-life extension was markedly lower than that of the positive control or the combination of DMDC and 10 ppm of natamycin (a ~ 0.05, Fig. 2B). Although the combination of DMDC and natamycin provided longer shelf-life

5 76 SIRICURURATANA ET AL. J. Food Prot., Vol. 76, No. 1 FIGURE 2. Microbial stability of cold-filled Concord and Niagara grape juices inoculated with low and high yeast levels and treated with various antimicrobial combinations. (A) Concord juice with low yeast inoculum; (B) Concord juice with high yeast inoculum; (C) Niagara juice with low yeast inoculum; (D) Niagara juice with high yeast inoculum. extension than did the individual compounds alone, the microbial counts almost reached the spoilage cutoff (10 6 CFU/ml) at the end of the study; thus this combination was not as effective as the positive control. In contrast, for the Niagara grape juice matrix, the use of DMDC alone or in combination with either concentration of natamycin was as effective as the traditional sorbatebenzoate treatments (a ~ 0.05). Each of these treatment regimens prevented spoilage in Niagara juice for up to 6 months, both at low and high yeast inoculation levels, representing cold packing of pasteurized and reconstituted juice or juice produced from fresh or frozen grapes (Figs. 1C, 1D, 2C, and 2D, respectively). The discrepancy in microbial counts toward the end of the storage period in high yeast inoculated Niagara juice that was treated with either DMDC or sorbate-benzoate (Fig. 1D) could be due to intersample variation. The low concentration of natamycin alone (10 ppm) extended the shelf life by up to only 56 days after low yeast inoculation, but the high concentration of natamycin (20 ppm) was as effective as sorbate-benzoate. This finding suggests that 10 ppm of natamycin is not sufficient to prevent yeast growth over an extended period of time when low concentrations of yeasts are present. For the natamycin-treated Niagara juice with either low or high yeast inoculation, yeasts were not detectable early in the storage period and then began to increase. As with the Concord juice, the inoculated yeasts were initially inhibited by natamycin but recovered and resumed growth during storage when the antimicrobial effect disappeared. When Niagara juice containing natamycin was challenged with the high yeast inoculation, shelf-life extension was reduced approximately two- to threefold, suggesting that natamycin at the concentration tested might not be sufficient to prevent yeast growth at high inoculation levels. The results observed for Concord and Niagara grape juice treated with natamycin are comparable to those in previous studies with pineapple juice preservation (14) in which addition of 10 and 20 ppm of natamycin delayed the growth of multiple strains of Saccharomyces cerevisiae (35 to more than 72 days) and Zygosaccharomyces bailii CRA229 (more than 72 days). The increased shelf life observed for Niagara juice compared with Concord juice when each was treated with DMDC alone or in combination with natamycin could be due to a synergistic effect of these antimicrobials and the residual free SO 2 originally presented in Niagara grape juice. In previous studies (4, 11, 14, 16), this synergic effect of DMDC and SO 2 has been reported in grape juices and wines. However, this hypothesis is not supported by the observations of Terrell et al. (14), who found that the combination of DMDC and SO 2 in Venus grape juice treated at 21uC was less effective for delaying fermentation than was treatment with DMDC alone. Dissolved CO 2 reduced the ph of carbonated juices (Table 2) and exerted an additional protective effect,

6 J. Food Prot., Vol. 76, No. 1 NATURAL ANTIMICROBIALS FOR GRAPE JUICE 77 FIGURE 3. Microbial stability of carbonated, cold-filled Concord (A) and Niagara (B) grape juices inoculated with high yeast levels and treated with various antimicrobials. thereby extending the shelf life even further than that observed for the still juices. Shelf-life extension by the DMDC plus natamycin treatment in carbonated Concord and Niagara juices was identical to that of the positive control and greater than that for similar treatments in still juices (a ~ 0.05, Fig. 3). In the comparison of Concord and Niagara juices, although the yeast counts in the positive control and the DMDC plus natamycin treatments of carbonated Concord juices never reached the spoilage cut off, the levels were notably higher than those in the Niagara juices (a ~ 0.05). The most promising result of this study is the identification of a universally efficacious protocol for increasing the shelf life of both still and carbonated coldfilled Niagara and Concord grape juice matrices to lengths comparable to those achieved with the traditional sorbate and benzoate additives (.161 days). The addition of 250 ppm of DMDC plus 5 to 10 ppm of natamycin effectively resulted in a stable Niagara grape juice product without the use of traditional chemical additives. However, even though this combination treatment provided shelf-life stability for Concord grape juices, it was not as effective as the sorbate plus benzoate treatment. The use of this DMDC preservative system warrants further investigation, but it has the potential to enhance the shelf life of these juices while appealing to consumers interested in beverages with natural antimicrobials. 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