788

Journal of Protection, Vol. 77, No. 5, 2014, Pages 788–795 doi:10.4315/0362-028X.JFP-13-362 Copyright G, International Association for Food Protection

Pickled Production: Effect of Brine Acetic Acid Concentration and Packing Conditions on Acidification Rate

OSCAR ACOSTA, XIAOFAN GAO, ELIZABETH K. SULLIVAN, AND OLGA I. PADILLA-ZAKOUR*

Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 West North Street, Geneva, New York 14456, USA

MS 13-362: Received 4 September 2013/Accepted 5 December 2013 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/5/788/1686987/0362-028x_jfp-13-362.pdf by guest on 27 September 2021

ABSTRACT U.S. federal regulations require that acidified must reach a pH of 4.6 or lower within 24 h of packaging or be kept refrigerated until then. Processes and formulations should be designed to satisfy this requirement, unless proper studies demonstrate the safety of other conditions. Our objective was to determine the effect of brine acetic acid concentration and packing conditions on the acidification rate of hard-boiled . Eggs were acidified (60/40 egg-to-brine ratio) at various conditions of brine temperature, heat treatment to filled jars, and postpacking temperature: (i) 25uC/none/25uC (cold fill), (ii) 25uC/none/2uC (cold fill/refrigerated), (iii) 85uC/none/25uC (hot fill), and (iv) 25uC/100uC for 16 min/25uC (water bath). Three brine concentrations were evaluated (7.5, 4.9, and 2.5% acetic acid) and egg pH values (whole, , four points within egg) were measured from 4 to 144 h, with eggs equilibrating at pH 3.8, 4.0, and 4.3, respectively. Experiments were conducted in triplicate, and effects were considered significant when P , 0.05. Multiple linear regression analysis was conducted to evaluate the effect on pH values at the center of the yolk. Regression analysis showed that brine concentration of 2.5% decreased the acidification rate, while packing conditions of the hot fill trial increased it. Inverse prediction was used to determine the time for the center of the yolk and the total yolk to reach a pH value of 4.6. These results demonstrate the importance of conducting acidification studies with proper pH measurements to determine safe conditions to manufacture commercially stable pickled eggs.

Eggs make important nutritional contributions to the mechanisms by which pH reduction of food allows the American diet (16), and research carried out during the last prevention of outgrowth of spores, reduction of heat resistance decade has shown that egg consumption does not of microorganisms, and decrease or inhibition of their growth correspond with an increase in coronary heart disease (as rate are mentioned in (5). once suggested), because there is no marked adverse effect Care must be taken so that the processing steps to on blood concentrations (7). Total egg produc- produce pickled eggs are carried out correctly to assure a safe, tion in the United States during 2011 was estimated at 91.9 stable, and acceptable product. In this sense, variations in acid billion, with 86% destined for table eggs (the rest used as concentrations or processing times may affect the safety and hatching eggs) (11). The majority of eggs destined for stability of the product if process conditions are inadequate. consumption are distributed to the food industry for use as Texture and flavor may be negatively affected if the product is ingredients in product formulation (4). Common technolo- over processed. An example of inadequate process conditions gies applied for increasing shelf life and/or assuring the of home-prepared pickled eggs is linked to a case of botulism safety of eggs include pasteurization, freezing, and dehy- (3), and at least one recall of pickled eggs has been reported, dration. Salting and alkaline have long been used as possibly due to contamination during manufacturing (18). On methods to preserve eggs (mostly duck eggs) and obtain the other hand, even though freshly laid eggs are generally products with particular characteristics (10, 6). Eggs are also sterile (9), and the thermal treatment applied to harden the egg commercialized hard boiled and preserved by packaging in is usually severe enough to inactivate potentially harmful modified atmospheres (4) or pickled. microorganisms (8), improper handling before the acidifica- Pickling is a long-standing method of preserving food, tion step (for instance, during the peeling operation) can which can be applied to various fruits and vegetables, as well contaminate the surface of the egg. as to foods of animal origin, such as eggs and meats. Federal regulations (21 CFR 114) indicate that to According to federal regulations (21 CFR 114), a pickled guarantee the safety of acidified foods, a thermal treatment product is an acidified food, which is a low-acid food to which should be applied for a sufficient time period to destroy the acid is added, its water activity is greater than 0.85, and it vegetative cells of microorganisms of public health signifi- has a finished equilibrium pH of 4.6 or below (20). The cance and those of nonhealth significance capable of reproducing in the food under normal conditions of storage, * Author for correspondence. Tel: 315-787-2255; Fax: 315-787-2284; distribution, retail, or use. Regulations also state that acidified E-mail: [email protected]. foods shall be manufactured, processed, and packaged so that J. Food Prot., Vol. 77, No. 5 EFFECT OF PACKING CONDITIONS ON ACIDIFICATION RATE OF PICKLED EGGS 789

TABLE 1. Experimental design for acidification of hard-boiled eggs with acetic acid (AA) brines at 60/40 egg-to-brine ratio Brine Heat treatment Postpacking Brine concn Trial temp (uC) to filled jars temp (uC) (%, wt/wt, AA)

Cold fill 25 None 25 7.5 4.9 2.5 Cold fill/refrigerated 25 None 2 7.5 4.9 2.5 Hot fill 85 None 25 7.5 4.9 2.5 Water bath 25 100uC/16 min 25 7.5 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/5/788/1686987/0362-028x_jfp-13-362.pdf by guest on 27 September 2021 4.9 2.5 a finished equilibrium pH value of 4.6 or lower is achieved total product are given): 7.5%/3%/3.8, 4.9%/2%/4.0, and 2.5%/ within the time designated in the scheduled process (20). 1%/4.3. The equilibrated target AA concentration was calculated Concerning this time period, regulations in 9 CFR 381 state using a mass balance that included the mass and concentration of that in an acidified low-acid product, every component must AA in the brine and eggs before acidification. Distilled white have a pH of 4.6 or lower within 24 h after the completion of vinegar (indicating 5% AA and purchased from a local supermarket) was used straight for the 4.9% brine, diluted by half the thermal process, unless data are available from the with distilled water for the 2.5% brine, or with added 99.7% establishment’s processing authority demonstrating that a glacial AA (VWR Scientific Products, West Chester, PA) for the longer time period is safe (19). Otherwise, the acidification 7.5% brine (28.2 ml/1,000 ml of vinegar). Sodium benzoate must take place under refrigerated conditions. Most resources (J. T. Baker, Phillipsburg, NJ) was added to the 2.5% AA brine to specify that home-prepared pickled eggs must be stored in the obtain a concentration of 0.05% preservative in the initial brine. refrigerator and not left at room temperature, other than The brine concentrations were confirmed by titration. during the period of time for serving (21, 12). Eggs differ from other products (such as most fruits and Preparation of samples. Four separate trials (each one vegetables), due to composition and size, which can affect including the three previously mentioned brine concentrations) the rate of acidification. The composition and structure of were carried out, as detailed in Table 1. For every trial, 946-ml glass jars with two-piece metal lids were used. Nine eggs were the yolk, which has a high content of and protein (32.5 placed in each jar, and the brines were added according to the and 17.5%, respectively) (14), can pose a challenge to quick conditions indicated in Table 1. Brines were added to obtain a and complete acidification. 60/40 egg-to-brine ratio (by weight). This ratio was determined by Although some studies have addressed acidification a preliminary trial to be the greatest egg-to-brine ratio, resulting rates of hard-boiled eggs (15, 1, 2), none refer to the in the top layer of eggs being completely covered with brine. variations in acid penetration due to the effects of a variety For the cold fill and cold fill/refrigerated trials, immediately of pickling acid strengths and processing conditions. Our after the brine was poured in the jars, the caps were tightened, and objective was to determine the effect of brine acetic acid the jars were tipped once to mix the brine around the eggs. No heat (AA) concentration and packing conditions (brine fill treatment was applied, and jars were stored at 25uC (cold fill) and temperature, heat treatment to filled jars, and postpacking 2uC (cold fill/refrigerated). For the hot fill trial, the brines were temperature) on the acidification rate of hard-boiled eggs. heated to 85uC and poured in the jars, which were immediately capped and tipped once. The jars were left to cool and stored at 25 C. For the water bath trial, the brines were poured in the jars, MATERIALS AND METHODS u the caps were tightened, and the jars were then preheated in a water Preparation of eggs and brines. Fresh, medium, grade A bath at 43uC for 5 min. The jars were then submerged in a water eggs were purchased from a local supermarket. Water was brought bath at 100uC and held for 16 min. The jars were left to cool and to a boil using an electric kettle (Groen TDB/6-10, Jackson, MS), then stored at 25uC. One of the eggs located in the middle layer in and eggs were laid in a wire basket in a single layer. Once a rolling the jar was pierced in the center with a flexible thermocouple, and boil was achieved, the wire basket of eggs was placed in the water, temperatures were registered at 10-s intervals during the thermal so the eggs were completely submerged. The eggs were boiled for treatment. 10 min (timing started once the water returned to a boil). After Each experiment was carried out in triplicate, and each jar cooking, eggs were immediately cooled in potable running water. represented a separate sample. After 4, 24, 48, 72, 96, and 144 h, Eggs were then stored at 2uC until samples were prepared. one egg was taken out from each jar, rinsed with distilled water, Immediately before preparing samples, eggs were warmed to 25uC and dried with a paper towel. A corresponding amount of brine was in a water bath and peeled by hand. removed to maintain the 60/40 egg-to-brine ratio in each jar and to For each trial, three brines of different concentrations of AA measure the brine pH at each sampling time. Eggs were cut were prepared. The characteristics of each brine are as follows longitudinally in half, and pH values were measured in four (initial AA concentration in the brine/equilibrated target AA sampling points of the egg as follows: the edge of white (point A), concentration in the total product/equilibrated final pH value of the halfway between the edge of the white and the edge of the yolk in 790 ACOSTA ET AL. J. Food Prot., Vol. 77, No. 5 the thickest part of the white (point B), the edge of the yolk adjacent to the thickest part of the white (point C), and the center of the yolk (point D). Afterwards, the yolk was ground, and pH was measured. Finally, ground yolk and white were combined and homogenized, and pH of the whole egg was measured. Hard-boiled eggs and eggs at the end of the pickling period processed following the conditions indicated in Table 1 (with the 7.5% AA brine) were analyzed for firmness. Eggs were cut longitudinally in half, and halves were placed with the cut side facing down, so the puncture probe would enter the outer surface of the egg. Three points were measured, corresponding to points A, B, and the edge of the white, closest to the yolk (between points B and C). The largest value of force (N) obtained from those three points was chosen as the representative value for that trial. Measurements of texture were conducted in triplicate. Whole Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/5/788/1686987/0362-028x_jfp-13-362.pdf by guest on 27 September 2021 pickled eggs and brines were sampled at the end of the experiment (after 144 h), and titratable acidity analyses were performed. Measurements were conducted in triplicate. FIGURE 1. Variation of pH with storage time at 25uC (cold fill trial, brine acetic acid concentration of 7.5%, 60/40 egg-to-brine Methods of analysis. pH was measured using an Accumet ratio). Error bars represent SD for n ~ 3. Dotted line marks the Basic AB15 pH meter (Fischer Scientific, Pittsburgh, PA), critical pH value of 4.6. Diagram of egg with sampling points is equipped with a calibrated Orion 8220BNWP micro ROSS also shown. glass-body pH electrode (Thermo Scientific, Beverly, MA). Titratable acidity was determined using a G20 compact titrator (Mettler Toledo, Schwerzenbach, Switzerland) and reported as correspond to , show fast diffusion of acid. percentage of AA (wt/wt). Egg firmness was determined at room Because point D (center of the yolk) is shown to have the temperature using the TA.XT2 Texture Analyzer (Texture slowest acidification rate, it was chosen as the representative Technologies Corporation, Scarsdale, NY). A puncture test was point to determine when the egg was thoroughly acidified, performed, with a puncture diameter of 3.32 mm, crosshead speed by use of multiple linear regression analysis. of 1 mm/s, and a travel distance of 4 mm. Figures 2 and 3 show a representation of variation of pH in sampling point D, total yolk, total egg, and brine, as Statistical analysis. A multiple linear regression analysis was a function of storage time, for every trial, at each one of carried out to evaluate the effects of storage time (after a the three tested brine concentrations. Consistently, higher logarithmic transformation), brine AA concentration, packing concentrations of AA in the brine increase the acidification conditions, and both their interactions with time, on pH values at the center of the yolk (point D). A random effect of replicate rate of the eggs. The total yolk measurements indicate it (nested within brine concentration and packing conditions) was acidifies faster than point D, while measurements of the also added. Effects were considered significant when P , 0.05. total egg show even faster acidification rates, due to the Acidification rates correspond to change in pH per change in contribution from the egg white (which acidifies the fastest). storage time (after a logarithmic transformation), thus lower and Although the effect of process conditions is not evident in higher rates would correspond to more positive and negative slopes most cases, the cold fill/refrigerated conditions show longer (respectively), because the linear relationship between pH and log- acidification times for every sampling point and brine AA transformed time during acidification is negative (pH decreases concentration. with time). Figure 4 shows a characteristic temperature-time profile Inverse prediction (which corresponds to the reverse process obtained from the thermal treatment applied to pickled eggs to linear regression) was used to predict the value of the processed according to the conditions of the water bath trial. independent variable (logarithmic transformation of process time) Jars were immersed in boiling water at minute 5 and from a given value of the dependent variable (pH at point D). This process allowed determination of the time (hours) when pH 4.6 removed at minute 21. Accumulated lethality values, shown was reached at the center of the yolk (point D) and on the total in Figure 4, were calculated every 10 min from the end of yolk, for every trial at each one of the three tested brine the immersion time. Accumulated lethalities (in minutes) concentrations. The 95% confidence intervals with respect to an were calculated using the reference values established for expected response were calculated. For the texture measurements, ensuring the commercial sterility of acidified foods, with a z means were compared using analysis of variance. Differences were ~ 8.9uC and reference temperature 93.3uC (13). If pickled considered significant when P , 0.05. Analyses were performed eggs were processed following the conditions described in using the statistical software JMP Pro 9.0.2 (SAS Institute Inc., this experiment, the thermal treatment corresponding to the Cary, NC). water bath trial (100uC for 16 min) would yield an appropriate lethality value to assure shelf stability of RESULTS products with a final equilibrated pH of 4.2 (or below). Figure 1 shows representative curves of variation of pH The observed lethality is well above one that corresponds to on different sampling points of the egg and brine, with a process of 2.5 min at a reference temperature of 93.3uC, storage time (conditions correspond to cold fill trial and the minimum required for a maximum equilibrium pH of brine AA concentration of 7.5%). Points A and B, which 4.2 (13). J. Food Prot., Vol. 77, No. 5 EFFECT OF PACKING CONDITIONS ON ACIDIFICATION RATE OF PICKLED EGGS 791

FIGURE 2. Variation of pH with storage time at sampling point D (N), total yolk (#), total egg (.), and brine (n), for the cold fill (25uC) and cold fill/refrigerated (2uC) trials, at the three tested brine acetic acid (AA) concentrations, 60/40 egg-to- brine ratio. Error bars represent SD for n ~ 3. Dotted line marks the critical pH value of 4.6. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/5/788/1686987/0362-028x_jfp-13-362.pdf by guest on 27 September 2021

The multiple linear regression analysis conducted on Table 3 shows the equilibrated target AA concentration pH values measured at point D (residual unexplained in the total product, as well as the measured AA replicate-to-replicate variability, ,7%) showed that the concentration in eggs and brines at the end of the pickling lowest brine concentration (2.5% AA) has a significantly experiments, for every trial and initial AA concentration in lower acidification rate (more positive slope) when the brine. Mean values and standard deviations (SD) for compared with the other two brine concentrations of 4.9 triplicate measurements are shown. In most cases brines and 7.5% (P , 0.05). Packing conditions of the hot fill trial show significantly higher titratable acidity values than significantly increased the acidification rate (more negative whole eggs, which indicates an incomplete equilibration of slope) (P , 0.05). In terms of intercepts, brine concentration acid concentration in the product. did not have a significant effect (P . 0.05), but packing A significant (P , 0.05) textural change was detected conditions did: cold fill/refrigerated and hot fill trials had when hard-boiled eggs (0.6 ¡ 0.1 N) were compared with significantly different (higher) intercepts (P . 0.05). These eggs at the end of the pickling period (1.0 ¡ 0.2 N). latter effects are likely caused by the influence of the first 4 h However, results showed that there were no significant of acidification (which were not monitored). differences in texture of egg samples subject to different Table 2 shows the calculated times to reach a pH of 4.6 process conditions at the end of the pickling period (P . at the center of the yolk (point D) and in the total yolk, for 0.05). These results indicate that texture is overall affected every trial at each one of the three tested brine concentra- by the pickling process but not by the method employed. tions. Averages and 95% confidence intervals with respect to an expected response are shown, and results were DISCUSSION obtained through inverse prediction from the fitted linear Federal regulations (21 CFR 114) specify that to relationship between pH and log time. In accordance with measure pH on food samples, such as pickled eggs, samples results in Figures 2 and 3, the cold fill/refrigerated must be prepared by draining the solids and blending them conditions show longer acidification times for every to a uniform, workable paste (20). This corresponds to the sampling point and brine AA concentration. Higher total egg sampling point, shown in Figure 1, which acidifies concentrations of AA in the brine appear to consistently at a faster rate than the center of the yolk (point D). decrease the times required for eggs to reach a pH of 4.6. Figures 2 and 3 show that for every trial condition, at initial 792 ACOSTA ET AL. J. Food Prot., Vol. 77, No. 5

FIGURE 3. Variation of pH with storage time at 25uC at sampling point D (N), total yolk (#), total egg (.), and brine (n), for the hot fill and water bath trials, at the three tested brine acetic acid (AA) concen- trations, 60/40 egg-to-brine ratio. Error bars represent SD for n ~ 3. Dotted line marks the critical pH value of 4.6. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/5/788/1686987/0362-028x_jfp-13-362.pdf by guest on 27 September 2021

brine concentrations of 7.5 and 4.9% AA, after just 4 h of concentration), the pH of whole egg samples were near or acidification, the whole egg samples have already achieved over 4.6 in every case. Therefore, one could underestimate pH values under 4.6, while samples from the center of the time needed to reach a pH of 4.6, if the pH of the whole the yolk have pH values well over the critical value of 4.6. egg is used as indicator. The faster acidification rate in the For eggs acidified with the 2.5% AA brines (initial whole egg (when compared with the center of the yolk or the whole yolk) was predictably observed in every brine AA concentration and packing condition tested throughout the experiment. Figures 2 and 3 also show the differences between acidification rates of eggs pickled in brines of different acid concentration. As will be further demonstrated by the multiple regression analysis, there are significant differences between the acidification rates of eggs pickled in brines of different acid concentration, and as expected, higher concentrations of AA produce a faster acidification of the eggs. The uneven acidification of eggs is most likely dependent on the composition (and structure after the egg is hardened by boiling) of the egg white and yolk: the egg white has a water content of 88%, while the yolk has only 48%. The 12% solids of egg white are virtually all protein, but the yolk has a high fat (32.5%) and protein (17.5%) content. Eggs contain about two parts white to one part yolk FIGURE 4. Heating profile of pickled eggs processed in boiling water bath. Jars were immersed in boiling water at minute 5 and by weight (14). As has been previously reported (15, 1), removed at minute 21. The accumulated lethality values (calcu- even though the white has a higher initial pH than the yolk, lated using the reference values of z ~ 8.9 and T ~ 93.3uC) the acidification rate is consistently faster in the white. achieved are shown, calculated every 10 min from the end of the Acton and Johnson (1) speculated that the penetration of immersion time. acid to the yolk is dependent on two factors: (i) diffusion J. Food Prot., Vol. 77, No. 5 EFFECT OF PACKING CONDITIONS ON ACIDIFICATION RATE OF PICKLED EGGS 793

TABLE 2. Calculated times for pickled eggs (60/40 egg-to-brine ratio) to reach a pH of 4.6 at the center of the yolk and in the total yolk, using inverse prediction from the fitted linear regressions Time (h)

Center of the yolk Total yolk Brine concn Trial (%, wt/wt, acetic acid) Avg 95% CI Avg 95% CI

Cold fill 7.5 23.5 19.7–27.5 8.4 6.1–10.8 4.9 34.4 29.2–40.2 10.2 7.1–13.4 2.5 75.9 61.6–96.8 32.1 25.9–39.3 Cold fill/refrigerated 7.5 56.5 47.2–68.5 24.3 20.0–28.8 4.9 82.7 67.4–105.1 36.4 30.9–42.8 2.5 220.3 154.9–359.2 89.1 74.8–109.1

Hot fill 7.5 30.6 25.8–35.9 10.5 7.4–13.8 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/5/788/1686987/0362-028x_jfp-13-362.pdf by guest on 27 September 2021 4.9 39.5 33.5–46.5 16.0 12.0–20.1 2.5 66.2 54.5–82.4 44.7 37.5–53.5 Water bath 7.5 26.0 22.6–29.5 1.4 0.4–2.8 4.9 38.1 33.3–43.6 5.1 2.7–8.0 2.5 93.7 76.9–118.4 30.1 23.2–38.1 rate of AA through the egg white and (ii) initial acid of 2.5% AA. As for the brine fill temperature, results concentration in the pickling medium. Manufacturers of showed that the acidification rate increased with the packing pickled eggs and similar products should determine a proper conditions of the hot fill trial, which mainly differs because way of establishing when the target pH in their products has of its higher initial temperature. Not many studies have been achieved, in compliance with regulations. The point addressed factors that affect acidification rates of pickled with the slowest acidification rate should be selected to eggs. Although Acton and Johnson (1) indicate that the monitor this change, and experiments should be conducted penetration of acid to the yolk is dependent on the initial according to the product and process characteristics. acid concentration in the pickling medium, their results Point D was consequently chosen to compare the showed no apparent difference between acid intake rates of acidification rates of pickled eggs processed with different pickled eggs brined with 3 and 5% AA vinegar solutions, brine AA concentrations and packing conditions (brine fill although no statistically sound method of comparison was temperature, heat treatment to filled jars, and postpacking mentioned. It must also be taken into consideration that the temperature). brines used in this experiment contained between 40 and Results from the multiple linear regression analysis 45% sucrose and 6% commercial pickling spice, which indicate that the most important factors that affect the could have reduced the effect of the acid strength on the acidification rate of pickled eggs are the brine concentration acidification rate. Although Ball and Saffores (2) also and the brine’s fill temperature. In the first case, the higher studied pickling of eggs in solutions with different acid brine AA concentrations (7.5 and 4.9%) decreased the egg strengths (ranging from 1 to 6% AA), they did not mention pH at a faster rate, compared with the lowest concentration differences in acidification rates when comparing the

TABLE 3. Equilibrated target acetic acid (AA) concentration in the total product (60/40 egg-to-brine ratio) and measured AA concentration in whole eggs and brines at the end of the experimenta

Titratable acidity (%, wt/wt, AA)

Trial Initial in the brine Equilibrated target in the total product Measured in whole egg Measured in brine

Cold fill 7.5 3.0 2.62 ¡ 0.04 2.98 ¡ 0.03 4.9 2.0 1.65 ¡ 0.01 1.80 ¡ 0.01 2.5 1.0 0.720 ¡ 0.009 0.759 ¡ 0.006 Cold fill/refrigerated 7.5 3.0 2.70 ¡ 0.04 3.01 ¡ 0.04 4.9 2.0 1.75 ¡ 0.03 1.87 ¡ 0.02 2.5 1.0 0.82 ¡ 0.01 0.788 ¡ 0.005 Hot fill 7.5 3.0 2.68 ¡ 0.03 2.97 ¡ 0.04 4.9 2.0 1.77 ¡ 0.03 1.86 ¡ 0.03 2.5 1.0 0.86 ¡ 0.03 0.776 ¡ 0.007 Water bath 7.5 3.0 2.59 ¡ 0.05 2.80 ¡ 0.03 4.9 2.0 1.66 ¡ 0.02 1.73 ¡ 0.01 2.5 1.0 0.730 ¡ 0.006 0.717 ¡ 0.003 a End of experiment, 144 h. Mean values ¡ SD for n ~ 3 are shown. 794 ACOSTA ET AL. J. Food Prot., Vol. 77, No. 5 different solutions. They only indicated that the pH of egg inoculated on the egg surface, thus reinforcing the need for white, yolk, and pickling solutions equilibrated after 6 to proper acidification studies to determine the most reliable 7 days at room temperature. conditions for reaching target pH and pathogen inactivation. A simpler analysis of the acidification data was Results of measured titratable acidity (percentage of performed by means of inverse prediction from the fitted AA) in the eggs and brines at the end of the pickling linear relationship between pH and log time, which allowed experiment (Table 3) show that more than 144 h are needed calculating the time when pH 4.6 was reached at point D to observe a complete equilibration of acid concentration in (center of the yolk) and in the total yolk, for every trial and the product, although this time is not considered as every brine concentration tested. Results in Table 2 show important as the time required to reach the critical pH of the marked differences between the cold fill/refrigerated 4.6 (or lower) in the center of the yolk. Predictably, most trial and the rest of the processing conditions (when values of measured titratable acidity in eggs and brines are comparing each one of the three brine concentrations), as lower than the equilibrated target AA concentration in the well as the differences between brines in every trial: higher total product, which is likely due to neutralization of acid by Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/5/788/1686987/0362-028x_jfp-13-362.pdf by guest on 27 September 2021 AA concentrations mean a lower time to reach the targeted compounds present mainly in the egg whites (14). pH. The relatively high predicted time of 220.3 h (and the Besides the concentration of acid in the brine and corresponding large confidence interval) for the 2.5% AA packing conditions, other process parameters should be brine at the cold fill/refrigerated trial occurs because after considered when assessing the acidification rate of hard- 144 h (final sampling point of the experiment), the center of boiled eggs. For instance, the ratio of eggs and brine will the yolk had barely reached a pH of 4.6. affect the acidification rate, because more acid will be Pickled egg processors might not be able to purchase available to enter the egg if the proportion of brine is higher. and use a microelectrode to determine pH in specific points The addition of other ingredients (such as salt or spices) of the yolk (such as the center). However, the pH of the could also affect the acidification rate. Acton and Johnson ground whole yolk can be determined easily with standard (1) found that during preparation of the pickle solution, a pH probes, and blending of semisolid products to a paste significant reduction of the AA concentration occurred (20 consistency is deemed an acceptable method of sample to 23% decrease), possibly due to the absorption and preparation for pH analysis (20). Data from Table 2 indicate neutralization of acid by the ingredients added to the vinegar that for pickled eggs manufactured following the described solutions (which include mustard, coriander, , process conditions and formulation (60/40 egg-to-brine ginger, allspice, dill seed, black pepper, bay leaves, cloves, ratio), every process (except for the cold fill/refrigerated chilies, and mace). conditions) at brine AA concentrations of 7.5 and 4.9% was The results discussed in the present study demonstrate able to produce pickled eggs that reached a pH of 4.6 the importance of conducting acidification studies with (measured on the total yolk) under 24 h. However, if AA proper pH measurements to determine safe conditions to concentration of 2.5% is used in the brine, the product manufacture commercially stable pickled eggs. These should be kept under refrigerated conditions until the whole results should be analyzed in context with validation and egg yolk reaches a pH of 4.6 or lower (approximately challenge studies that address the survival of pathogenic 4 days), unless data are available from a processing microorganisms in pickled egg systems, with the ultimate authority demonstrating that a longer time at room goal of establishing science-based recommendations that temperature to reach pH 4.6 is safe (19). can be used by the food industry. Given that acidification of the center of the yolk occurs at a slower rate when compared with the whole yolk, data from REFERENCES Table 2 also show that no process condition was able to 1. Acton, J. C., and M. G. Johnson. 1973. Pickled eggs. 1. pH, rate of produce pickled eggs that presented a pH value of 4.6 acid penetration into egg components and bacteriological analyses. (measured in the center of the yolk) in less than 24 h (using Poult. 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