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Fertilized chick eggs were incubated horizontally (Day 0) without rotation (Fig. 1a). On Day 3 of incubation, eggs were cracked and the yolk and its associated embryo were quickly laid into a weighing boat (Fig. 1b and Video S1). Embryos with a beating heart and an intact underlying yolk were considered as healthy, whilst eggs with a leaking yolk, damaged blood vessels or an unfertilized appearance were discarded. Eggs with a healthy appearance were transferred in their weighing boat to an internally humidified chamber, made up by a plastic box containing 50 ml sterilized H2O and closed with a transparent lid (Fig. 1c and Video S1). The transparent lid allowed for daily observations of embryonic development without the need to open the internal humidified chamber, which minimized infection and improved survival.
Fertilized white Lohman and Bovan chick eggs were obtained from Strömbäcks Ägg, Vännäs, Sweden. The use of fertilized chick eggs prior to E14 does not require an ethical permission. The chick eggs were incubated horizontally (day 0) in an egg incubator (Fiem) without rotation in relative humidity of 70% and temperature of 37.5 C. On Day 3 of incubation the eggs were sprayed with 70% ethanol and left to dry at room temperature (RT). Egg was cracked and the yolk and its attached embryo were transferred to a weighing boat (VWR International). The weighing boat was placed in a 0.4 L plastic box (Esclain) containing 50 ml sterilized H2O and closed with a transparent lid, creating an internally humidified chamber, in which embryos were further incubated.
Century eggs are one of the traditional delicacies originating from China. This research investigated the difference between traditional and industrial methods of making century eggs, through various factors such as the number of steps required and time required for century eggs to complete their formation. Secondary to this comparison, the chemical process and scientific theory behind the formation of these eggs was explored. The traditional method involved wrapping a batch of eggs in a chemical clay and leaving them for 10 days. The industrial method, however, involved soaking the eggs in a chemical solution for a while. Both methods observed century egg formation in which the albumen proteins denature under an alkaline condition. However, the stated 10 day period of the traditional method was not enough to completely preserve the eggs. Future experiments could include chemically dissolving the eggshells in advance to reduce the diffusion barrier and distance for the alkaline conditions to reach the albumin of the egg. In turn, this would reduce the time required for century eggs to form, meaning that production costs could be lowered.
The high pH causes the protein to denature breaking down the interactions between the amino acids in the secondary and tertiary structures (the secondary structure is the initial folding of the amino acid sequence into an alpha helix or a beta-pleated sheet and tertiary structure is the further folding of the protein to make a 3D arrangement)[3]. This leads to the proteins in the albumen (egg white) to solidify and, since water is present, gelatinise. The strong alkaline conditions also break down fats through a process known as saponification, this leads to the development of a unique flavour for the century egg [4]. At the same time, the metal ions, as well as tannins (a class of chemicals that bind to and precipitate proteins) found in the black tea, prompt the protein to solidify [5]. Consequently, the proteins such as those in the albumen are broken down into different amino acids, such as threonine, isoleucine and leucine. These amino acids further produce hydrogen, ammonia and a very small quantity of hydrogen sulphide. Ammonia and hydrogen sulphide, which are well-known for their pungent smell, are essential to provide flavours for the century eggs [1]. Furthermore, the amino acids would take salt form under alkaline conditions, resulting in the formation of crystals, with the shapes of pines, in the albumen of a century egg. Another main source of protein in the egg is the yolk, which is high in sulphur. As the yolk proteins are broken down through the action of hydroxyl ions, they produce hydrogen disulphide and hydrogen. The pigments in the egg yolk are combined with all kinds of metal ions, which leads to the dark green colour of the yolk [1].
A feed solution was made using a combination of chemicals in which the duck eggs were placed into (see Table 4 in the Appendix). This involved mixing sodium carbonate and black tea powder to the bottom of each container, before adding boiled water and calcium oxide. Each container with the feed solution was mixed thoroughly until all the contents dissolved. 25ml of the surface layer from each container was extracted and placed into a mortar. Zinc chloride was added and grounded into the mortar, before this mixture was added back into the containers. The solutions were then allowed to rest for three hours at the end of which sodium chloride was added. After a further 24 hours of sitting, the solutions were mixed thoroughly again and the duck eggs were split between the containers (see Fig. 13 in the Appendix) for 45 days. The three containers were covered with a layer of gloves filled with water to fully submerge the eggs. Chicken eggs were also placed into the solution for comparison to the duck eggs. Due to a lack of funding and storage issues, the size of the experiment was scaled down. Ideally, all duck eggs should be placed in one container filled with the feed solution. However, there was great difficulty in finding a large enough container to fit all the eggs in so splitting up of the solution and eggs between three containers allowed the eggs to be easily transported if there was any need to change the place of storage[8].
This experiment involved making a feed clay that was used to wrap the eggs in (see Table 5 in Appendix for ingredient list). Calcium oxide, sodium carbonate, plant ash (a by-product of burning charcoal), sodium chloride and tea leaves were added together. Water was then added and all chemicals were mixed thoroughly to form the clay-like mixture. The clay was left for 24 hours before it was used to wrap each egg and rolled in rice hulls so a layer covered the outside of the clay mixture (see Fig. 14 and Fig. 15 in the Appendix) All 12 containers were sealed (see Fig. 16 in the Appendix) for a 10-day duration. One egg was cracked open every day to note any observations. One of the 12 containers contained only chicken eggs which were only opened at the end of the 10 days. Another contained only duck eggs for the same purpose. The other 10 containers had a few duck eggs and one chicken egg indicated by a blue ink mark in each[9].
At the end of the industrial method, a few eggs were randomly picked out of the batch to be opened and examined. After 45 days, five eggs were opened and four of the eggs completely formed century eggs, as shown in Fig. 1. As figure 1 has shown, the albumen of the century eggs has turned into a black-green colour and has gelatinized. The yolk which Fig. 1 does not show has also turned dark green and solidified. Even though one of the eggs had not fully solidified, all five produced the distinctive smell and flavour of a century egg. Likely, this egg was not a long way from completion.
Overall, the 10 days allowed for the eggs to be wrapped in the feed clay did not show much progression in century egg formation. Throughout the 10 days, five eggs were opened every day to check for progress and see how they changed. The layer of clay wrapped around each egg was removed and each egg was washed with water.
Five eggs were cracked open but there were no changes observed to the albumen and the yolk (see Fig. 2), which was the expected outcome. Both were still liquid-like and the yolk still yellow. Also, there was no distinctive smell produced.
As the eggs were being washed, the eggshells felt like they were covered by a layer of membrane. However, the inside of the duck eggs had no obvious difference to the previous days. Both the yolk and the albumen were still liquid-like and the yolk specifically still yellow, as seen in Fig. 6.
When the eggshells were cracked open, the top layer of the yolk had started to solidify, with all of them beginning to turn green. The yolk was still liquid-like below the layer, but it had turned into a golden colour, as shown in Fig. 7. A weak smell unique to a century egg was produced by all five eggs. Some sedimentation of the gelatinised egg yolk was observed on the eggshell (see Fig. 17 in the Appendix).
The major difference between the traditional and industrial method was the time factor. The industrial method was completed over a duration of 45 days to allow the eggs to be left in an inaccessible place. Whereas, the traditional method was completed over a suggested duration of 10 days and is likely the reason for the large difference in stage of century egg formation observed [9]. However, only those eggs in the last 2 days showed unexpectedly slow progression towards completion, so this may be due to problems that only posed to the eggs and conditions in these two batches. One possible explanation could be that these containers were not fully airtight, but to conclusively show this would require further investigation.
The eggs from the industrial method were not able to be sent to a laboratory for composition analysis so only the composition of the eggs of the traditional method could be discussed. The eggs did not completely form into century eggs using the suggested traditional method and therefore the composition was compared to a store-bought century egg to see if the stage of formation the eggs were at could be concluded [9]. This can be seen from Table 2 as there is a noticeable difference between the duck century egg produced through the traditional method and the century egg bought on the market. This could mean that the century eggs produced were still not close to completion. Since the eggs in the traditional method were not completed during the 10 days, it can not be said which method would have taken longer. Furthermore, the eggs in the industrial method were only opened after 45 days, meaning they could have been completed at an earlier date. Without a mathematical or scientific technique to extrapolate the results in the traditional method, it could not be determined which method would have taken longer for completion. Another plausible explanation would be due to the individual compositional differences between eggs. Since the differences shown in Table 2 are not very large and not all the composition differences fall between both the bought duck egg and century egg, this explanation may be more likely.