Effect of basic alkali-pickling conditions on the production of lysinoalanine in preserved eggs

Effect of basic alkali-pickling conditions on the production of lysinoalanine in preserved eggs Yan Zhao,,, Xuying Luo,, Jianke Li,, Mingsheng Xu, and Yonggang Tu,1 Jiangxi Key Laboratory of Natural Products and Functional Food, Jiangxi Agricultural University, Nanchang 330045, China; Engineering Research Center of Biomass Conversion, Ministry of Education, Nanchang University, Nanchang 330047, China; and State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China ABSTRACT During the pickling process, strong alkali causes significant lysinoalanine (LAL) formation in preserved eggs, which may reduce the nutritional value of the proteins and result in a potential hazard to human health. In this study, the impacts of the alkali treatment conditions on the production of LAL in preserved eggs were investigated. Preserved eggs were prepared using different times and temperatures, and alkali-pickling solutions with different types and concentrations of alkali and metal salts, and the corresponding LAL contents were measured. The results showed the following: during the pickling period of the preserved egg, the content of LAL in the egg white first rapidly increased and then slowly increased; the content of LAL in the egg yolk continued to increase significantly. During the aging period, the levels of LAL in both egg white and egg yolk slowly increased. The amounts of LAL in the preserved eggs were not significantly different at temperatures between 20 and 25 o C. At higher pickling temperatures, the LAL content in the preserved eggs increased. With the increase of alkali concentration in the alkali-pickling solution, the LAL content in the egg white and egg yolk showed an overall trend of an initial increase followed by a slight decrease. The content of LAL produced in preserved eggs treated with KOH was lower than in those treated with NaOH. NaCl and KCl produced no significant effects on the production of LAL in the preserved eggs. With increasing amounts of heavy metal salts, the LAL content in the preserved eggs first decreased and then increased. The LAL content generated in the CuSO 4 group was lower than that in either the ZnSO 4 or PbO groups. Key words: preserved egg, lysinoalanine, alkali-pickling condition, metal ions 2015 Poultry Science 94:2272 2279 http://dx.doi.org/10.3382/ps/pev184 INTRODUCTION Preserved egg is a traditional Chinese delicacy. Preserved egg white is a brown or greenish brown gel. There are lots of crystals which looks like pine needles on the surface and interior of preserved egg white, so preserved eggs are also known as Songhua (Chinese) eggs. The egg yolk can be shades of blackish green, grass green, or dark brown and b of their beautiful color, the clear crystals, high nutritional value, and unique flavor, preserved eggs are popular with consumers in China, East Asia, and Southeast Asia. The processing of preserved egg has a history extending back more than 600 years in China. The eggs can be prepared by pickling fresh eggs in a solution of water, alkali, salt, Chinese black tea, and metal ions at room temperature for 30 to 40 days (Su and Lin, 1993; Wang and Fung, 1996). The basic principle of preserved egg C 2015 Poultry Science Association Inc. Received January 21, 2015. Accepted May 29, 2015. 1 Corresponding author: tygzy1212@aliyun.com processing is to use alkali to denature and coagulate the proteins in the eggs (Wang and Fung, 1996) and thus the alkali plays a critical role in converting a fresh egg into a preserved egg. Heavy metal ions also play an important role in controlling the penetration speed of the alkali from the pickling solution into the egg, as well as promoting the coagulation of the proteins (Wang and Fung, 1996; Tu et al., 2013). Salt adjusts the flavor of the preserved eggs, accelerate egg white degeneration and solidification, and facilitates the separation of the eggshell (Zhao et al., 2010; Zhao et al., 2014). Today s formulations for preserved egg processing include alkali, heavy metal salts, and salt. NaOH and NaCl are the most common alkali and salt used in the preparation of preserved eggs, although KOH and KCl are also sometimes used to produce low-sodium preserved eggs (Zhang et al., 2011; Wang et al., 2013). Given the danger of Pb to human health, copper salts (CuSO 4 ), zinc salts (ZnSO 4 ), or mixed copper and zinc salts are now generally used to replace PbO in the preserved egg process (Ganasen and Benjakul, 2010; Ganesan and Benjakul, 2010). The pickling temperature affects the penetration rate of the alkali into the eggs, thereby 2272

PRODUCTION OF LYSINOALANINE IN PRESERVED EGG 2273 affecting the quality and production cycle of preserved eggs (Sun et al., 2011). Currently, investigations of the alkali-pickling conditions for preserved eggs mainly aim to shorten the pickling cycle, or to improve egg quality and stability as well as to reduce the accumulation of heavy metals and other hazards. However, the safety of preserved eggs should not be limited to the hazard represented by heavy metals, which aspect has received much attention. LAL, a potentially hazardous chemical generated in preserved eggs should also be addressed. LAL, which has a chemical name of N ɛ -(DL-2-amino- 2-carboxyethyl)-L-lysine, was first discovered in alkali (ph 13) treated bovine pancreas ribonuclease A by Bohak in 1964 (Bohak, 1964). High temperature and alkali treatment can promote the production of LAL in protein-rich foods (Finley and Kohler, 1979; Miller et al., 1983; Hasegawa et al., 1987; Faist et al., 2000; Calabrese et al., 2009). The formation of LAL not only reduces the nutritional value and digestibility of the food, but also decreases the utilization of minerals in the body by chelating many metallic elements to inactivate the metalloenzymes (Hayashi, 1982). It can cause kidney diseases in mice (Leegwater, 1978; Karayiannis et al., 1979). Although toxicity of LAL to human body is not yet clearly understood, because of its potential harmfulness and before its toxicology is proven, its content should be strictly controlled in food, and especially in food for infants (D Agostina et al., 2003). This study has investigated the effects of basic alkalipickling conditions on the production of LAL in preserved eggs in order to provide a reference for controlling the production of LAL in the preserved eggs process. Material MATERIALS AND METHODS LAL was obtained from Bachem (Bubendorf, Switzerland). DL-2, 6-diaminopimelic acid, triethylamine, N-methyl-N-(tert-butyldimethylsilyl)trifluoro acetamide, N,N-dimethylformamide, and ovalbumin were purchased from Sigma-Aldrich (St. Louis, MO). Analytical grade HCl, NaOH, CuSO 4, NaCl, PbO, KOH, and ZnSO 4 were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). High purity water for the dilution of concentrated hydrochloric acid was obtained from a Mill-Q purification system (Millipore, Bedford, MA). of laying from a farm in Nanchang County, Jiangxi Province, China. Duck eggs were cleaned with tap water and checked for any cracks prior to pickling. Forty clean duck eggs were soaked in pickling solutions containing 4% NaOH, 4% NaCl, and 0.4% CuSO 4 for 30 days at constant temperature (25 o C) (Tu et al., 2013). The eggs were removed from the alkali-pickling solution after 30 days, the solution on the surface of eggs was removed, and the eggs were then aged at 25 o C for 18 days. To test different pickling and aging times, starting from the first day, the preserved eggs were sampled every 6 days during the processes. After checking the sensory characteristics, the egg white and egg yolk were separated and stored at 20 o C for later use. Preparation of Preserved Eggs Under Different Pickling Temperatures. Fresh duck eggs were preserved with alkali-pickling solution containing 4% NaOH, 4% NaCl, and 0.4% CuSO 4 for 30 days at 20 o C, 25 o C, 30 o C, and 35 o C. After 30 days, the preserved eggs were sampled. The egg white and egg yolk were separated and stored at 20 o C for later use. Preparation of Preserved Eggs Under Different Types and Amounts of Alkalis. Solutions of 3%, 3.5%, 4%, 4.5%, 5%, and 5.5% NaOH or KOH were prepared containing 4% NaCl and 0.4% CuSO 4, respectively (Zhang et al., 2015). The fresh duck eggs were preserved at 25 o C for 30 days. After 30 days, the preserved eggs were sampled. After checking the sensory characteristics, the egg white and egg yolk were separated and stored at 20 o C for later use. Preparation of Preserved Eggs with Different Amounts of NaCl and KCl. Solutions of 0, 1%, 2%, 4%, 8%, and 16% NaCl or KCl were prepared containing 4% NaOH and 0.4% CuSO 4, respectively. The fresh duck eggs were preserved at 25 o C for 30 days. After 30 days, the preserved eggs were sampled. After checking the sensory characteristics, the egg white and egg yolk were separated and stored at 20 o C for later use. Preparation of Preserved Eggs with Different Types and Amounts of Heavy Metal Salts. Solutions of 0, 0.05%, 0.1%, 0.2%, 0.4%, and 0.8% CuSO 4, ZnSO 4, or PbO or a 1:1 mixture of ZnSO 4 and CuSO 4, were prepared containing 4% NaOH and 4% NaCl, respectively (Tu et al., 2013). The fresh duck eggs were preserved at 25 o C for 30 days. After 30 days, the preserved eggs were sampled. After checking the sensory characteristics, the egg white and egg yolk were separated and stored at 20 o C for later use. METHODS Preparation of Preserved Eggs Preparation of Preserved Eggs with Various Pickling and Aging Times. Fresh duck eggs with weight of between 65 and 70 g were obtained within 5 days Test of the Sensory Characteristics of the Preserved Eggs After the alkali-pickling solution on the surface was removed, the preserved eggs were air dried and carefully peeled to check how easily they could be separated from

2274 ZHAO ET AL. Figure 1. Changes of LAL content in the preserved eggs during the pickling and aging periods. the shell. The egg white and egg yolk were then separated. The color, state of solidification, hardness, and elasticity of the egg white, as well as the color, state of solidification, and the size of the watery center of the egg yolk were assessed (Ma, 2007). Measurement of LAL Intact preserved eggs were selected for LAL detection. The LAL content was measured using the analysis method previously established by our research group (Luo et al., 2013). Data Statistics and Analysis All experiments were performed in triplicate and results expressed as means ± SD (n = 3). Duncan s multiple comparison of ANOVA was conducted with the IBM SPSS Statistics 19 software, and P < 0.05 was considered statistically significant. RESULTS AND DISCUSSION Effects of the Pickling Time and Aging Time on the LAL Production in Preserved Eggs The LAL levels in the egg white and egg yolk of the preserved eggs were measured. The results (Figure 1) showed that LAL was not detected in the egg white or egg yolk of the fresh eggs. With increased pickling time, the LAL contents in the egg white and egg yolk of the preserved eggs reached 2047.91 ± 37.57 mg/kg and 1094.88 ± 38.20 mg/kg, respectively, after 30 days and 2096.03 ± 56.56 mg/k 2 g and 1162.86 ± 23.79 mg/kg, respectively, after 18 days of aging. The changes of LAL concentration during the processes of pickling and aging are shown in Figure 1. Throughout the processes of pickling and aging, the changing patterns of LAL content in the egg white and egg yolk showed some differences. During the 30 days of the pickling period, the LAL produced in the egg white rapidly increased during days 0 to 12 (P < 0.05) and increased at a slower rate during days 12 to 30; the LAL produced in the egg yolk continued to increase markedly during the entire pickling period (P < 0.05). During the 18 days of the aging period, the changes of LAL generated in the egg white and egg yolk were not significant. In addition, comparison of samples from different time points showed that the amount of LAL generated in the egg white was higher than that in the egg yolk. The formation of lysinoalanine in food with a high protein content under alkali treatment may occur via a 2-step reaction: OH, SH or S S functional groups of serine, cysteine, and cystine are heterolyzed under alkali conditions, resulting in a β-elimination reaction and the formation of dehydroalanine that contains a conjugated double bond. A nucleophilic addition reaction occurs between the double bond of dehydroalanine and ɛ-nh 2 of lysine, resulting in the formation of LAL (Maga, 1984; Friedman, 1999). The formation of LAL is affected by factors including protein source, ph, temperature, and time. Higher ph values and temperatures, and longer times are more favorable to the formation of LAL (Friedman et al., 1984; Chang et al., 1999). The protein source, including the type and concentration of proteins, is one of the major factors affecting the production of LAL. The egg white contains approximately 9.7 to 10.6% proteins, including ovalbumin, ovotransferrin, and ovomucoid; the egg yolk contains approximately 15.7 to 16.6% proteins, including low-density lipoprotein, yolk globulin, phosvitin, and high-density lipoprotein (Kovacs-Nolan et al., 2005). Although the protein content in egg white is lower than that in egg yolk, the LAL content generated in the egg white was higher because ph is another important factor affecting the production of LAL in preserved eggs processed with alkali. During the process of pickling at a constant temperature, NaOH penetration into the egg is continuous. It first enters the egg white and then the egg yolk. During the early stages of pickling, the ph of the egg white increases rapidly; during days 8 to 12, the ph of the egg white reaches a maximum and then decreases slightly. During the entire preservation period, the ph of the egg yolk continues to increase, but reaches a value lower than that of the egg white (Zhang, 2005; Yang et al., 2012). During the pickling process, increased levels of LAL are produced by the rapid ph increase in the egg white and egg yolk and by extending the pickling time. During the aging period, the amount of LAL in the egg white and egg yolk slightly increases, and this then is affected only by the extension of time. Therefore, ph change is an important factor driving the changes of LAL present during the pickling process and aging period.

PRODUCTION OF LYSINOALANINE IN PRESERVED EGG 2275 Figure 2. Effect of pickling temperature on the LAL content generated in preserved eggs. Effects of Temperature on LAL Production in Preserved Eggs The sensory quality of the preserved eggs showed that the pickling temperature influenced the sensory quality of preserved eggs. Preserved eggs can be prepared at temperatures of 20 to 35 o C. The preserved eggs white prepared at 20 o Cand25 o C were a translucent greenishbrown, with good shell separability and excellent flexibility and hardness; the egg yolk had a solidified layer of approximately 3 to 4 mm, and the outer layer was dark green with multi-colored rings and a low viscosity large watery center. For the preserved eggs prepared at 30 o C and 35 o C, the egg white was a translucent brown, with poor shell separability and a sticky shell (especially for the 35 o C group), and the elasticity was less than that of the first 2 groups, with greater local hardness and a softened small head ; the egg yolk had a solidified layer of approximately 4 to 5 mm, and the outer layer was dark green, with multi-colored rings and a small watery center. The pickling temperature has a clear impact on the levels of LAL generated in the egg white and egg yolk of the preserved eggs (Figure 2). The LAL contents generated were not significantly different (P > 0.05) between the egg white and egg yolk for the eggs prepared at 20 o Cand25 o C. The LAL contents in the egg yolk for the eggs prepared at 30 o Cand35 o C were also very similar (P > 0.05). However, generally speaking, increasing the pickling temperature increased the amount of LAL generated in both the egg white and the egg yolk of the preserved eggs. The processing of preserved eggs works on the principle that alkali and other substances in the alkali-pickling solution enter the egg white and then the egg yolk through pores in the eggshell and eggshell membrane, and through corrosion holes generated by the alkali, resulting in the denaturation and coagulation of the proteins in the eggs (Ma, 2007). The alkaline substances in the alkali-pickling solution enter the egg white through holes in the eggshell and shell membrane to increase the concentration of alkali in the egg white. The alkaline substances that enter the egg white from the alkali-pickling solution also permeate into the egg yolk, which reduces the alkali concentration in the egg white; therefore, at a constant temperature, the alkalinity of the egg white is undergoing a dynamic process with an increase followed by a decrease (Zhang, 2005). Elevating the temperature can accelerate the penetration of the alkaline substances from the alkali-pickling solution into the egg white and then the egg yolk, thereby increasing the alkalinity of the egg white and egg yolk (Sun et al., 2011). Therefore, the LAL produced in the egg white and egg yolk of preserved eggs increases with increasing temperature. This pattern is more obvious in the egg yolk because higher temperatures accelerate the penetration of the alkaline substances in the alkali-pickling solution, accelerating the coagulation of the egg yolk and resulting in a lower moisture content in the egg yolk (Sun et al., 2011); thus, the amount of LAL produced per unit mass of yolk shows a more obvious increasing trend. Effects of the Type and Amount of Alkali on the LAL Production in Preserved Eggs The test of the sensory quality of preserved eggs prepared with different amounts of NaOH or KOH showed that 3% and 3.5% KOH were the only conditions that could not produce preserved eggs. Among all the preserved eggs, the sensory quality of those prepared with 4%, 4.5%, 5% NaOH, or 5%, 5.5% KOH was relatively normal. The impact of the amount of alkali on the LAL content generated in the egg white and egg yolk in the preserved eggs prepared with NaOH or KOH are shown in Figure 3. As shown in Figure 3a, with an increase in the mass fraction of NaOH in the alkali-pickling solution, the LAL content generated in the egg white and egg yolk first increased and then slightly decreased. Figure 3b shows that with an increase in the mass fraction of KOH in the alkali-pickling solution, the LAL content generated in the egg white first increased and then slightly decreased, whereas the LAL content generated in the egg yolk continued to increase. The LAL content generated in the egg white and egg yolk increased with increases in the mass fraction of NaOH or KOH in the alkali-pickling solution, caused by the increased alkalinity (or ph) in the egg white and egg yolk. However, the content of LAL also showed a slight decrease with an increase in the mass fraction of the base in the alkali-pickling solution, which may be because the ph in the egg white and egg yolk was too high and the generated LAL was slightly decomposed after a long period of preservation. When the mass

2276 ZHAO ET AL. Figure 3. Effect of the amount of alkali on the LAL content generated in preserved eggs (A. NaOH, B. KOH). fractions of NaOH and KOH in the alkali-pickling solution were equal, the amount of LAL produced in both egg white and egg yolk in eggs preserved with KOH were lower than in eggs preserved with NaOH. This result is consistent with the finding of Chu et al. (1976) who reported that the amount of LAL generated in corn treated with KOH is lower than in corn treated with NaOH. This result demonstrates that although KOH and NaOH are both strong alkalis, they perform differently in catalyzing the formation of LAL. Effects of NaCl and KCl on the LAL Production in the Preserved Eggs Various mass fractions of NaCl and KCl were added into the alkali-pickling solution of the preserved eggs, and a sensory quality test was conducted after 30 days. The impacts of NaCl and KCl on the sensory quality of the egg white and egg yolk were similar. With NaCl or KCl mass fractions increasing from 0 to 8%, the shell separability improved, and the watery center in the yolk gradually shrank. Further increasing the mass fractions of NaCl or KCl to 16% prevented the coagulation of the egg white and hardened the egg yolk. The impacts of different mass fractions of NaCl or KCl on the LAL content generated in the egg white and egg yolk of the preserved eggs are shown in Figure 4. For preserved eggs prepared with 0 to 16% NaCl and KCl in the alkali-pickling solution, there are no significant differences in the egg whites among the various concentration groups (P > 0.05). However, when the mass fraction of NaCl continued to increase from 8 to 16%, and KCl continued to increase to 16%, the amounts of LAL generated in the egg yolk increased significantly (P < 0.05). For the preserved eggs prepared with equal mass fractions of NaCl and KCl, the amount of LAL generated in the egg white or egg yolk were not significantly different (P > 0.05), indicating that the impacts of these 2 salts on the LAL produced in the preserved eggs were not significantly different. Adding NaCl or KCl in different mass fractions to the alkali-pickling solution caused no significant difference (P > 0.05) in the amount of LAL generated in the egg white of the preserved eggs. However, as the mass fraction increased, the amount of LAL generated in the egg yolk also increased because the NaCl and KCl in the alkali-pickling solution generated a concentration gradient between the two sides of the semipermeable eggshell membrane and the yolk inner membrane, resulting in an osmotic pressure. This osmotic pressure promoted a continuous penetration of the NaCl or KCl from the alkali-pickling solution through the membrane toward the egg white and then the egg yolk, causing the rupture and gelation of the yolk granules. The free water in the egg white directly diffused into the alkali-pickling solution, and the free water in the egg yolk diffused outwards into the egg white, and then the alkali-pickling solution, and thus the egg yolk hardened (Chi and Tseng, 1998;Ma,2007). Although both the egg white and the egg yolk underwent dehydration, when the amount of NaCl or KCl was higher, the dehydration of the egg yolk was greater (Lai et al., 1999), and thus the relative protein concentration in the egg yolk increased, resulting in an increase of the LAL content per unit of yolk. These results suggest that the choice of NaCl or KCl may have no effect on LAL production in the preserved egg. Effects of Heavy Metal Salts on LAL Production in Preserved Eggs Different types and concentrations of heavy metal salts produced various effects on the formation and sensory quality of the preserved eggs. In general, under the alkali-pickling conditions of the present study, CuSO 4

PRODUCTION OF LYSINOALANINE IN PRESERVED EGG 2277 Figure 4. Effect of NaCl and KCl on the LAL generated in preserved eggs (A. NaCl, B. KCl). is the most favorable in terms of egg quality, followed by the mixed salt of CuSO 4 and ZnSO 4 in a mass ratio of 1:1, whilst the performances of ZnSO 4 and PbO were poor, with unstable quality, poor shell separability, and a high chance of rotten end and liquefaction at the sharp end in the preserved eggs (Ma, 2007; Tu et al., 2013). In addition, different concentrations of the same type of heavy metal salt may result in different sensory qualities of the preserved eggs. Within the mass fraction range used in the present study, only 0.8% ZnSO 4 and 0.8% PbO failed to produce stable preserved eggs. The addition of 0.1 to 0.4% CuSO 4, 0.2 to 0.4% ZnSO 4, ZnSO 4 and CuSO 4 mixed salt with a total mass fraction of 0.4%, or 0.4% PbO to the alkali-pickling solution resulted in a normal sensory quality of the egg white and egg yolk in the preserved eggs. As an auxiliary material for egg preservation, CuSO 4 in different mass fractions generated different amounts of LAL in the egg white and egg yolk. As shown in Figure 5a, as the mass fraction of CuSO 4 in the alkalipickling solution increased from 0.05 to 0.8%, the levels of LAL generated in the egg white and egg yolk of the preserved egg first decreased and then increased. Heavy metal ions such as Cu 2+ can chelate with lysine via its ɛ-nh 2, and this can limit the generation of LAL in the preserved eggs by reducing the concentration of the substrate involved in the formation of LAL (Friedman, 1999). During the process of egg preservation, heavy metal ions can regulate the entrance of alkali into the egg during late stages of egg preservation by plugging the hole (Ma et al., 2001; Zhao et al., 2010), and this can also reduce the generation of LAL in the egg white and egg yolk. Therefore, CuSO 4 can reduce or partially inhibit the generation of LAL in the egg white and egg yolk of the preserved eggs. Theoretically, this inhibition is enhanced by an increase in the mass fraction of CuSO 4 in the alkali-pickling solution. In reality, when the mass fraction of CuSO 4 increased to 0.8%, the LAL content in both the egg white and the egg yolk rebounded slightly, and this phenomenon requires further investigation. Because 0.8% ZnSO 4 could not produce stable preserved eggs, the amount of LAL in the egg white and egg yolk were only quantified for preserved eggs prepared with auxiliary material ZnSO 4 at a mass fraction of 0.05 to 0.4%, and the results are shown in Figure 5b. WithanincreaseinthemassfractionofZnSO 4 in the alkali-pickling solution from 0.05 to 0.4%, the LAL content generated in the egg white and egg yolk of the preserved eggs continued to decrease. In this study, the mixed salt of CuSO 4 and ZnSO 4 in a mass ratio of 1:1 was used as an auxiliary material for egg preservation. The impact of the total mass fraction of the mixed salt in the alkali-pickling solution on the LAL content generated in the egg white and egg yolk of preserved eggs is shown in Figure 5c. With an increase in the total mass fraction of CuSO 4 and ZnSO 4 mixed salt in the alkali-pickling solution from 0.05 to 0.8%, the levels of LAL produced in the egg whites and egg yolks in the preserved eggs first decreased and then increased, which is similar to the results for preserved eggs prepared with CuSO 4 only as the auxiliary material. The regulatory effect of ZnSO 4 and CuSO 4 mixed salt on the LAL production in the egg whites and egg yolks of the preserved eggs is related to the chelation of these 2 metal ions with lysine and their ability to plug holes during the egg preservation process. However, mixed salt in a large mass fraction also promoted abnormal LAL production in the preserved eggs, which is similar to the results obtained from pickling with CuSO 4 alone. Because 0.8% PbO failed to produce stable preserved eggs, the amounts of LAL in the egg whites and egg yolks were only quantified for preserved eggs prepared with auxiliary PbO at mass fractions of 0.05 to 0.4%, and these results are shown in Figure 5d.Withanincrease in the mass fraction of PbO in the alkali-pickling solution from 0.05 to 0.4%, the LAL content generated

2278 ZHAO ET AL. Figure 5. Effect of heavy metal salts on the LAL production in preserved eggs (A.CuSO 4, B. ZnSO 4,C.CuSO 4 and ZnSO 4 (1:1), D. PbO). in the egg whites and egg yolks of the preserved eggs continued to decrease, which may be due to the chelation of PbO with lysine, as well as its ability to plug holes during egg preservation. The comparison of the LAL content in the preserved eggs prepared with various heavy metal salts showed that when the mass fraction was the same, the LAL contents in the egg whites and egg yolks were lowest in the preserved eggs prepared with CuSO 4 alone as the auxiliary material. CONCLUSION The basic conditions of alkali pickling have a strong impact on the production of LAL in preserved eggs. The basic alkali-pickling conditions should be controlled according to KOH 5 to 5.5%, NaCl 2 to 4%, and heavy metal salt CuSO 4 0.4%. The pickling temperature should be 20 to 25 o C, and the processing time should be kept as short as possible to control the amount of LAL produced, while obtaining preserved eggs with a normal sensory quality. ACKNOWLEDGMENTS This study was financially supported by the Program of National Natural Science Foundation of China (No.:31101293, 31101321, 31360398 and 31460400). REFERENCES Bohak, Z. 1964. Nɛ-(dl-2-Amino-2-carboxyethyl)-l-lysine, a new amino acid formed on alkaline treatment of proteins. J. Biol. Chem. 239:2878 2887. Calabrese, M. G., G. Mamone, S. Caira, P. Ferranti, and F. Addeo. 2009. Quantitation of lysinoalanine in dairy products by liquid chromatography mass spectrometry with selective ion monitoring. Food Chem. 116:799 805. Chang, H., C. Tsai, and C. Li. 1999. Inhibition of lysinoalanine formation in alkali-pickled duck egg (Pidan). Food Res. Int. 32:559 563. Chi, S.-p., and K.-h. Tseng. 1998. Physicochemical properties of salted pickled yolks from duck and chicken eggs. J. Food Sci. 63:27 30.

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