Physicochemical properties of NEW
Obtained neutral electrolyzed water physicochemical properties included pH 6.83±0.48, ORP (mV) 858±7.34 and chlorine content (ppm) of 58.85±2.48. It has been reported that neutral pH range is 6.4 to 7.5 18. The oxide-reduction potential value is consistent with the same study. The total chlorine concentration was similar to previous reports 12,19 considering the evaluated solution as a neutral electrolyzed water (EW).
Egg quality
Haugh units decreases with older hens and factors like temperature and storage time negatively affect egg quality 20. Obtained results for pH, percentage of total solids and Haugh Units are reported in Table 2.
Results showed that pH value was at the upper limit of the range specified by the Mexican Norm NMX-FF-127-SCFI-2016 21, and the percentage of content solids was high. As for Haugh Units and weight, values corresponded to egg quality Category II (Mexico) 21 or B quality (USDA) 1.
Liquid egg
Alpha amylase test
The alpha amylase test was performed to evaluate the efficiency of pasteurization process. Obtained results were compared to untreated egg where the enzyme was still present in the egg. Pasteurized liquid egg showed absence of α-amylase in the egg product (due to heat inactivation). Every liquid egg batch was confirmed by alpha amylase test before further use.
In vitro microbiology analysis
Evaluation of NEW at different temperatures
This test was carried out to know if temperature affects the bactericidal effect of NEW, because production of liquid egg products includes thermal process and, high temperature could generate some change in the solution that is evaluated in this study. The temperatures of 60°C and 80°C were selected because the pasteurization temperature of liquid whole egg is in this range and results were compared with those obtained at room temperature. According to Figure 1a, it was determined that for the three evaluated temperatures, the final count of microorganisms was below 1x103 CFU/mL when the inoculum was 1x108 CFU/mL demonstrating that NEW was able to decrease up to five exponents of Escherichia coli O157: H7 at rt, 60°C and 80°C reaching a 99.999% of bacterial reduction count, therefore, it was found that temperature was not a factor that influences the bactericidal effect of NEW.
Bactericidal evaluation at different concentrations
Antibacterial activity of NEW was evaluated against Escherichia coli O157: H7 at different NEW´s concentrations, since the solution when subsequently evaluated in liquid egg products will suffer a dilution effect. Results (Figure 1b) exhibited that bactericidal effect depended on hypochlorous acid concentration. When 10 ppm and 25 ppm concentrations were used, bacterial survival numbers reached values of 6.29 CFU/mL and 3.55 CFU/mL respectively. We detected a proportional correlation (r=-0.812) between bacterial reduction and NEW concentration, meaning that the higher NEW concentration, the greater the percentage of bacterial reduction. When 25 and 10 ppm were used, there was no significant difference between both concentrations.
In situ microbiology analysis
Bactericidal evaluation at different concentrations
Bactericidal NEW effect was evaluated in different liquid ovoproducts due to inner composition could affect bactericidal solution properties. At the same time, it is important to detect a concentration that kept bactericidal effect without affecting the minimum concentration of total solids in whole egg (24.4%), egg whites (12.13%) and yolk (51.30%) 22–24.
NEW was evaluated in contaminated liquid whole egg. Significant differences were detected in all evaluated concentrations (Figure 2a) when compared with SS treatment. However, there was no significant difference between 1, 2.5 and 5 ppm. When 25 ppm and 10 ppm were used, they were significant different from the rest of used concentrations. The percentage of bacterial reduction were 27.53%, 24.75%, 39.65%, 59.09% and 64.65% for 1ppm, 2.5ppm, 5 ppm, 10 ppm and 25 ppm respectively.
When NEW was used in contaminated whole egg, the percentages of reduction were lower than in vitro study; this effect caused by the presence of organic material was reported previously 25, where food proteins have sulfhydryl groups in their structure, and hypochlorous acid reacts on them at the same time as on those of the bacterial membrane.
For egg whites, the bacterial reduction percentage were 53.70% (2.5 X 104 CFU/mL) and 48.17% (2.8 X 104 CFU/mL) for 5 ppm and 2.5 ppm respectively (Figure 2b) and for contaminated egg yolks that were treated with NEW at 2.5 ppm and 5 ppm showed a bacterial decrease of 65.08% (1.03 X 105 CFU/mL) and 89.15% (3.0 X 104 CFU/mL) respectively. When contaminated yolks were treated (Figure 2c), it showed a significant difference when NEW was used at 5 ppm and the 2.5 ppm showed no statistical difference.
Regarding percentage reduction, it was observed that for whole egg and liquid whites showed lower bactericidal effect than when it was evaluated in liquid yolks. This effect could be attributed to the fact that yolk composition does not present a high protein content that could interfere with NEW bactericidal effect.
pH in different liquid egg products
NEW treatment was added using different concentrations to liquid whole egg (LWE) and pH value was monitored (Figure 3). The pH of LWE with NEW at 1 ppm and 2.5 ppm were significantly different from the rest of the treatments, however this difference was small because the pH difference is around 0.04. Likewise, the solution when interacting with the organic matter of the product does not release species with an acidic or basic character that contribute the pH change.
These characteristics were considered and pH value of treated LWE was monitored for three weeks, and no significant difference was detected when comparing 2.5 ppm and 5 ppm of NEW concentration with untreated egg (Figure 4a). It was interesting because egg composition presents changes in storage such as protein decarboxylation causing an increase in pH, however NEW had no retarding effect on that change.
For liquid whites (Figure 4b), it was observed that only at day 0 there was a difference with respect to control without treatment, however, for the following days this difference disappeared. In general, an increase in pH was observed, which was due to the breakage of peptide bonds.
Treated liquid yolks showed a pattern where pH decreased significant at day 14 and then it raised at day 21, showing no interference by treatments with this pattern. However, day 14 pH decrease was small (~0.1) (Figure 4c).
Color
Luminosity
Treated WLE with different concentration of evaluated solutions showed significant difference with respect to the sample that did not receive treatment (Figure 5). Differences were attributed to dilution effect, that was carried out in each sample; however, the group treated with SS at the same dilution, did not showed significant difference with respect to the control, except when SS was used at 2.5 ppm. It is known that NEW is an oxidizing substance, acts on lipid pigments like carotenoids, and lutein and zeaxanthin are the main pigments in eggs.
When the evaluation was measured over time (Figure 6a), a trend was detected to decrease L* parameter over the weeks. The luminosity changed immediately after the addition of NEW; nevertheless, this could occur due to the dilution effect. Subsequently, it was observed that treated samples with NEW did not present significant differences with respect to the controls, this indicates that the solution did not subsequently affect the luminosity of the whole egg and that the decrease in this parameter is not attributed to any treatment.
Luminosity was decreasing in treated and non-treated egg whites over time (Figure 6b). This effect was attributed to the high protein content and proteolytic activity by endogenous enzymes causing a decrease in viscosity and greater passage of light 26. These factors caused ferric sulfide formation which causes dark coloration causing a decrease in luminosity. This effect was not altered using NEW or SS.
When egg yolks were treated Figure 6c, all treatments generated an increase in luminosity, this increase was not detected in yolks without treatments. This increase was attributed as a yolk dilution effect.
Parameter a*
The parameter a* is the one that indicates the change of coloration from red (+) to green (-), in Figure 7, the control samples treated with SS as well as the samples treated with NEW at different concentrations showed significant difference with respect to the sample without treatment, in both samples an almost constant decrease in red coloration is observed. It is observed that the samples treated with NEW were those that presented less red coloration. This could be caused by oxidizing properties reaction with egg pigments, such as lutein and zeaxatin that have yellow-orange colorations, affecting its structure and with it, the ability to impact color.
When the study was evaluated over time (Figure 8a), treated whole egg presented lower values of red coloration with respect to the sample without treatment. We observed that NaCl 5 ppm solution does not caused this behavior, so the decrease in coloration is attributed to oxidation properties of NEW acting with egg pigments, causing less intense tones.
For egg whites (Figure 8b), the beginning of the monitoring it was observed that the samples presented a value of 0 on the color scale. This could be caused because egg whites are composed by proteins and water and there is no molecule that could provide coloration; however, it was observed that the samples treated with NEW and NaCl 5 ppm showed a more negative trend than the other samples.
Treatment with NEW and SS caused the yolks to have less red coloration (Figure 8c) because yolk was diluted, however at day 7 after treatment, significant differences were observed with respect to the control without treatment.
Parameter b*
Whole egg was treated with different concentrations of NEW and b* parameter was measured. This indicates yellowish (+) and blueish (-) colorations. NEW treatment caused decrease in yellow coloration (Figure 9) and the higher the concentration of NEW, the less yellowing. This effect is due by the presence of oxidizing species which interact with the carotenoids present in the egg. It is known that one of the factors that influences the decomposition of these is oxygen, generating structural changes (trans to cis) 5 which causes greater absorbance in the visible spectrum and therefore a slight shift in the observed color.
When whole egg was evaluated over time (Figure 10a) results showed that NEW treatment caused significance difference after 14 days of storage. The tendency of this for parameter was to decrease with respect to the storage time indicating loss of yellow coloration. Egg pigments or carotenoids, like xanthophylls, lutein, and zeaxanthin 27 are susceptible to degradation by storage and oxygen exposure. This promotes autoxidation of unsaturated molecules such as carotenoids. However, NEW did not significantly affect the pigment structure.
When liquid egg whites were treated and analyzed over time (Figure 10b), yellow coloration was not affected by the addition of NEW since there were no significant difference. This same behavior was observed for control samples with SS.
For treated liquid yolks (Figure 10c), NEW treatment did not modify the yellow-blue coloration and changes in the samples since a significant difference was only found at the beginning of the study, however, this could be due to the oxidation of carotenoids.
Delta E
Whole egg (Figure 11) was treated with NEW and total change color, defined as ΔE, was calculated. Electrolyzed water was evaluated at different concentrations, and it caused a difference in color change with respect to the sample that did not receive treatment, this result was attributed to the oxidant property of the solution which generated changes in the composition of the molecules.
NEW treatment was evaluated using two concentrations and whole liquid egg color evaluations were taken over time (Figure 12a). Both concentrations showed a similar pattern to NaCl treated group, showing a slight decrease in red color because of oxidation of pigments. Treated samples with NEW showed less color change than NaCl solution and this could be affected by the contribution of a* parameter, indicating a slight decrease in red color by effect of pigment oxidation.
For liquid white eggs (Figure 12b), treated groups presented the lowest color impact; this effect was due to the fact that there are not pigments in its composition and the small values changes were due to storage, likewise it was observed that NEW did not influence the color variation.
Finally, when yolks were treated (Figure 12c), the greater change in color was reached at day 0, however this differential was diminished over time. Luminosity was the main parameter affected by treatments, which could be affected by dilution process and a* was affected by possible oxidation of carotenoids. However, NEW group showed the less color change between all evaluated groups.
Emulsion capacity
The addition of NEW in liquid whole egg at 1 ppm, 2.5 ppm, 5 ppm and 10 ppm does not significantly affect the formation of the emulsion, however at a concentration of 25 ppm NEW, egg´s functional property was diminished, This behavior was mainly attributed to the fact that protein and phospholipids concentrations were in smaller quantity (by dilution effect) so it was not possible to emulsify a high amount of oil as in the other evaluated concentrations. It was also observed that emulsion capacity was low when SS was used at 5, 10 and 25 ppm (Figure 13).
As it was mentioned above, egg emulsion capacity ws given by the presence of phospholipids and lipoproteins to emulsify oil. Monitoring emulsion over time showed that on day zero no significant difference was found between the samples treated with NEW with respect to the sample without treatment or with NaCl (Figure 14a), however for day 7 of monitoring a loss of property was observed in all samples. After, at day 14, there was an increase in emulsion capacity and again no difference was found with respect to control treatments however, emulsion capacity was statistical lower than at day 0.
On day 21, the trend of the 14th was maintained. It can be observed that the samples treated with NEW and NaCl with 5 ppm presented a slight decrease however, differences were not significantly different between treatments at day 21 and this difference could be attributed to the loss of protein by storage.
For egg yolks (Figure 14b), there was a tendency to decrease the emulsion capacity. At day 0 of monitoring, emulsion capacity was decreased when NaCl at 5 ppm was used, no differences were found in NEW with respect to control treatment. However, at day seven after treatment, a statistical decrease in this capacity was detected and the lowest value by the group without treatment. Decreased emulsion capacity was kept for the rest of the monitoring time.
Foaming capacity
Foaming capacity is an important factor that is used in food processing. Proteins such as ovomucin and globulins are responsible for stabilizing and forming protein interaction that is capable of air retaining due to their amphipathicity. LWE was treated with NEW using different concentrations which did not show significant difference. Same pattern was detected when NaCl was used. This capacity was attributed to the fact that the addition of liquid is favorable for foam generation. However, the resulting foam tends not to be stable when NaCl was used (Figure 15).
When treatments were analyzed over time in LWE, the foaming capacity was not affected, except for day 14 where NEW (5 ppm) increased enhanced foaming when it was compared to the group without treatment (Figure 16a).
Treated liquid whites kept foam capacity after seven days of storage (Figure16b). NEW (5 ppm) and NaCl (2.5 ppm) showed the highest value. This performance was decrease at day 14 and it was kept until the last check point.
Acid content
Acid content was evaluated for the three egg products however, oleic and carbonic acid content was measured in whole egg and only oleic acid was measured in yolks since this acid has the greatest proportion. Oleic acid is an unsaturated fatty acid, and it was expected that after contact with NEW, it would be affected by oxidation reaction that would affect its concentration over time. For egg whites, carbonic acid was detected, and values were reported as percentage.
For LWE it was detected a decrease in acid concentration over time (Figure 17a) however, no significant decrease was detected. The only significant difference was between NEW treatment and NaCl 5 ppm at day 7 of storage where the NaCl showed the lowest acid content, and it was similar to no treatment value at day 21 of storage.
When liquid egg white was evaluated (Figure 17b), it was detected a decrease in acid content over time. Values were stable after seven and 14 days of storage and finally, all values dropped after 21 of storage. For the no treated group, values were stable at day zero and seven of storage and then dropped significantly. If we corelate pH results with the percentage of acidity, it was observed that both trends were inversely proportional because, as mentioned previously, during storage, proteins that compose egg albumen are susceptible to loss peptide bonds and increased pH due to exposure of amino ends. That explains that samples treated with NEW and NaCl did not present differences with respect to the control without treatment, so that the acidity variations were not attributed to evaluated solutions and they helped to keep acid content after 14 days of storage.
Finally, for egg yolks (Figure 17c) acid content was slightly decreasing over time and there were no variations between treatments. Possibly there were losses of oleic acid however this could be caused by storage.
Lipid oxidation (TBARS)
Whole egg was treated with two different NEW concentrations (Figure 18a) where MDA did not exceed 0.5 mg MDA/kg of sample at day 0, coinciding with previous studies where MDA values were 0.32 to 0.42 mg/kg of sample 28.
NEW is an oxidizing solution and it caused significant difference at day 21 when it was used at the highest concentration. These differences could be due to the loss of malonaldehyde which was able to react with free amino acids such as lysine, histidine, arginine tyrosine and methionine, causing loss of egg nutritional value 29.
The yolk is the main part that concentrate different types of egg lipids. The obtained data at day 0 showed that MDA value was low, Bernal et al (2003)30 reported that MDA concentration in fresh yolk was ~0.18 mg / kg coinciding with our results. Over time oxidizing activity caused that MDA tended to increase and the highest value was detected when NEW 5 ppm was used (Figure 18b).