Effects of Individual and Block Freezing on the Quality of Paciﬁc Oyster ( Crassostrea gigas ) during Storage under Different Pretreatment Conditions

: In this study, a series of pretreatments, including ice-glazing, polyphosphate impregnated, and both ice-glazing and polyphosphate impregnated, were employed to pretreat shucked oysters in order to explore the optimal processing conditions for long-time storage. The effect of repeated freezing-thawing cycles on the quality of oysters was evaluated. Several quality indicators were used to investigate the effects of pretreatment. For the VBN (volatile salt-based nitrogen) value, the lowest value was 9.1 ± 0.2 of BPG (block oyster with polyphosphate impregnated and ice-glazing), which was signiﬁcantly lower than 9.6 ± 0.2 of IPG (individual oyster with polyphosphate impregnated and ice-glazing). In terms of drip loss, there was no signiﬁcant difference between the IPG (21.0 ± 0.2%) and the BPG (20.8 ± 0.2%). In addition, the highest WHC% (water holding capacity) was IPG (65.5 ± 0.5%) which was slightly lower than BPG (67.6 ± 0.6%). As compared to the experimental control, the IPG and BPG had the best appearance and color. In terms of TAPC (total aerobic plate count), with the increase of freezing storage time, each group showed a slight downward trend, but the difference was not statistically signiﬁcant. After repeated freezing-thawing of block frozen oysters, there were signiﬁcant differences in drip loss, WHC, and cooked taste with the increasing number of times, and there was a trend of deterioration ( p < 0.05). Repeated freezing and thawing can seriously degrade the quality of oysters, so individual freezing (especially IPG) should be the most appropriate processing method.


Introduction
Pacific oysters (Crassostrea gigas) are rich in free and unsaturated fatty acids, with EPA (Eicosapentaenoic acid) and DHA (Docosahexanoic acid) levels of up to 14.5% and 9.5%, respectively. The amount of plant sterols (phytosterols) can be as high as 33% [1]. It also contains oyster peptides (bioactive peptides from oysters) which are an important part of bioactive peptides in humans. Aside from the protein, eight essential amino acids, taurine, vitamins, and trace elements such as zinc, selenium, iron, copper, and iodine are also abundantly contained in it [2,3].
According to the Food and Agriculture Organization of the United Nations, 2020 [4], oyster production reached 5.8 million metric tons worldwide in 2018, of which Taiwan's oyster production averages only about 35,000 metric tons per year. However, the demand for oyster meat in the Taiwan market is about 80,000 tons a year. The insufficient supply, which is dominated by frozen oysters, means most of them rely on imports. Thus, the methods we use to manage oysters prior to trading, such as transporting, cleaning, and storing them, as well as how we maintain, monitor, and keep them at a high level of nutrient and safety, have not only been critical issues for the food industry, but also the time and effort we have put into them.
Due to diversified nutrition, high moisture content, high microbial adhesion, strong enzyme activity, packaging type, processing technology, temperature difference, and change during storage, aquatic food is the main reason for the quality decline [5]. Common ones include lipid oxidation, protein degradation, microbial proliferation, and massive loss of drips, changes in appearance or color, etc. [6][7][8][9]. In order to improve the quality and preservation period of oysters, many scholars have put forward many valuable research results [10][11][12][13].
In the research of packaging content, some studies proposed using different proportions of gas components to package oysters. The experiment showed that the use of pure N 2 gas packaging under anoxic conditions had the best effect on prolonging the freshkeeping period [14]. According to additional research, using bags with antibacterial and anti-corrosion coatings can extend the freshness of oysters [15]. Furthermore, Cao et al. stated in their research report that the lower the freezing temperature, the fresher the oysters [16]. At the same time, Cao's research team also found that by means of combining ozone water with chitosan, the lifetime of oysters would last for two days at low temperatures of 5 • C in storage [17]. Regarding drip loss of frozen oysters, the study by Songsaeng et al., noted that individual quick freezing (IQF) has less drip loss than contact metal plate freezer (CPF), and IQF caused damage to the tissue of the oyster that was relatively minor and has a better appearance [18]. On the other hand, the liquid loss of oysters in ice storage was related to the salt content, and except for oysters with low salt content, most containers of oyster meats did not exceed 15% free liquid during storage [19]. More studies have pointed out that if oysters are depurated before freezing, the total number of bacteria can be effectively reduced by 96.7%, and the fresh-keeping period of oysters can be extended [20].
Indeed, shucked oyster meat is easy to contaminate due to the essence of its texture-soft and fleshy-which makes it a better habitat for microorganisms to grow if improper storage is adopted, eventually leading to a decline in quality and freshness. Manousaridis et al. and Hernández et al. successively demonstrated that a significant effect of sterilization could be achieved by treating oysters with ozone water [21,22]. Recently, many studies have used acidic electrolyzed water to depurate oysters and found that the bactericidal effect is very good [23,24].
Oysters have a large market in China, Taiwan, the Philippines, Vietnam, Thailand, etc., and are favored by consumers. Since the shucked oyster meat is soft and easy to break, it is not easy to clean. Therefore, the microorganisms attached to the oyster meat grow easily and multiply during the storage period, resulting in the loss of freshness and quality. For freshness preservation, the most common ones are ice-water refrigeration (freshwater or seawater), ozone treatment, freezing, etc. In traditional markets or supermarkets, it is mostly sold with soaked oyster meat or bagged soaked oyster meat, but it can only keep the freshness for three to five days; therefore, freezing the oysters should be an option if you want to keep the freshness for a long time. If the oysters are frozen, the quality may be degraded by some factors such as temperature, temperature change, and repeated freezing-thawing times during the frozen storage period [18,25].
The main reasons for causing the quality of oysters to reduce, in conjunction with the data collected from above statistically, fall on fat oxidation and protein degradation during storage, a large amount of drip loss after thawing; and the rapid growth of microorganisms. As for improving or maintaining freshness, most use ice glazing, adding antioxidants or phosphates, etc. [19,26]. In addition, there is very little information on the block and individual frozen oysters, so our team conducted research on the pre-treatment methods of oyster processing. The main appeals are: using ice-glazing, soaking phosphates, and implementing individual freezing and block freezing to explore whether the quality and preservation period of oysters can be improved. Among them, taking into account the cooking profile of consumers, it may be necessary to repeatedly freeze and thaw (especially block-type freezing) due to the large number of purchases, which may cause the risk of low quality. Therefore, repeated freezing and thawing of block frozen oysters were also added to the research framework to observe the changes in their quality.

Oyster Meat Treatments
Fresh shucked oyster meats were purchased from Baishuihu, Dongshi Township, Chiayi County, Taiwan. After initially cleaning with seawater, the oysters were placed in a cooler and immediately transported to the laboratory, then rinsed with distilled water, sorted, and placed on a stainless-steel screen for 15 min to drain excess water prior to dividing them into individually frozen oyster meat (IFOM), block frozen oyster meat (BFOM), and block frozen oyster meat repeated freezing and thawing (BFOM-R). Three groups were further split up, respectively, into four subgroups: According to the experiment, the required quantities of oysters are: individual frozen oysters, the quantity of each group is about 1.2 kg, 4 groups are 4.8 kg, these groups were placed on a stainless steel tray (2 × 20 × 45 cm) piece by piece and then frozen at −45 • C for 24 h; block frozen oysters, the quantity of each group is 4.8 kg (0.6 kg/box × 8 boxes), 4 groups total 19.2 kg; block freezing repeated freezing and thawing group, the quantity of each group is 2.4 kg (0.6 kg/box × 4 boxes), 4 groups total 9.6 kg. These groups were packed into a plastic box (4 × 12 × 18 cm) and then frozen at −45 • C for 24 h. Polyphosphate impregnated subgroups were soaked in 0.5% sodium triphosphate solution [27] for 2 h, then, scooped up and put on the stainless-steel screen for 15 min to drop dry. Subgroups treated with ice-glazing were soaked in ice-cold water for 5 s repeating 3 times. Lastly, all subgroups were placed into plastic bags, separately, and stored at −25 • C for up to 12 months. On average, the rate of ice-glazing for IFOM was 18.5%, whereas BFOM took up about 18.3%. Marked codes of 8 types of oyster meats were displayed in Table 1. During each step of the experiment, only a small portion from subgroups was thawed and sampled for the analyses. In addition, the repeated freezing-thawing of block freezing oyster meat is to thaw the frozen oysters that have been pretreated in a low temperature environment, and then freeze again immediately after the measurement was completed.

Chemical Compositions Analyses
In accordance with the methods 950.46, 940.25, and 991.36 of AOAC (2000) [28], analyses of moisture, crude protein, crude fat, and carbohydrates, etc., of the sampled oysters were subjected to analysis.

Thawing of Oysters
In advance of experimenting, the frozen oyster meats were taken out of each subgroup and put into a low-temperature refrigerator at a temperature of 5-7 • C for 24 h for thawing.

Determination of Volatile Basic Nitrogen
Volatile basic nitrogen (VBN) was measured using the micro-diffusion method [29]. Oyster meat (10 g) was homogenized with 20 mL of 7% trichloroacetic acid. The homogenate was centrifuged (8000 rpm, 10 min) and filtered with Whatman no. 1 filter paper. The volume of the filtrate was made to 100 mL with 7% trichloroacetic acid. An aliquot of 1 mL TCA extract and 1 mL saturated sodium carbonate was placed in the outer chamber of a Conway's dish. An aliquot of 1 mL 10% boric acid solution was put in the inner chamber of a Conway's dish. Afterwards, the Conway's dish was incubated at 37 • C for 90 min and the boric acid was titrated with 0.02 N HCl until light red. A control using 1 mL 7% trichloroacetic acid to replace oyster meat extraction was included for calculation. The VBN of oyster meat was calculated using the following equation: A: titration volume (mL) of 0.02 N HCl for the oysters B: titration volume (mL) of 0.02 N HCl for the blank F: 0.02 N HCl titer

pH Measurement
Five grams of oyster meat from each subgroup were added to 5 times the amount of distilled water and homogenized at 12,000 rpm at ambient temperature. The pH values of the homogenate were measured using a pH meter (LAQUA F-70 pH meter, HORIBA, Kyoto, Japan) [30].

Determination of Total Aerobic Plate Count (TAPC)
The determination of total aerobic plate count was conducted according to AOAC (1995a) [31] and Maturin et al. [32]. Oyster meat (25 g) was homogenized with 225 mL of sterile 0.85% sodium chloride. Serial dilution was made, and the pour plate method was used with plate count agar (Merck, Germany). Colonies were counted after being incubated at 37 • C for 48 h.

Oyster Color Attributes
The color analysis was conducted based on the method reported by Mridula et al. [33], with modifications. The color attributes of oyster meat were measured using a color meter (NE 4000, Nippon Denshoku Industries Co., Ltd., Tokyo, Japan), and the results were expressed as an a* value in the CIELAB color space. The a* value represented the values of the redness/greenness of samples, respectively. The standard whiteboard (Y = 93.06, X = 95.06, and Z = 112.04) was used for calibration, and the analysis was conducted in the reflectance mode. The thawed oyster was placed on the stainless-steel screen for 5 min to drain excess water, and then put on the quartz sample plate and measured. At least ten oysters were selected from each subgroup.

Determination of Drip Loss
The operation of the experiment adopted the method of Hasegawa [34] and Mousakhani-Ganjeh et al. [35] with some minor modifications. The thawed oyster was weighed (W 2 ) and the drip loss was calculated against the initial weight (W 1 ).

Water Holding Capacity Determination
Abided by the practice of Mousakhani-Ganjeh et al. [35] and Tironi et al. [36], with minor modifications. After thawing at 5 • C and drying the surface with filter paper, sampled oysters were then cut equally into segments, each weighing about 3 g (M 1 ). Next, each segment was wrapped in filter paper and centrifuged for 10 min (4000 rpm/min, 5 • C), and weighed again (M 2 ). The water holding capacity (WHC)% was calculated as

Sensory Evaluation
The sensory evaluation of repeatedly frozen-thawed oysters was carried out by 11 panelists of staff from the Department of Seafood Science, National Kaohsiung University of Science and Technology, using the modified guidelines from Cao et al. [16]; Songsaeng et al. [18]; and Lee et al. [37] with a scale from 1 to 9: 1-unacceptable; 2-extremely poor; 3-very poor; 4-poor; 5-acceptable; 6-good; 7-very good; 8-extremely good; and 9-excellent. The panelists were asked to score the intensity of each characteristic, describing appearance (shape, size) and taste (chewing elasticity, sweet, and refreshing).

Statistical Analysis
The data gained from two trials (three replicates for each trial) was subjective to the analysis of variance (ANOVA) in conformity with the experimental design in one-way factorial (4 freezing methods including 2 glazing methods and 2 impregnating methods) by the multivariate method. Comparison of means was accomplished by LSD and Scheffe's test for significant differences at p < 0.05.

Volatile Salt-Based Nitrogen (VBN)
As mentioned by Javier et al. [38], it was speculated that the reason for the VBN value increasing with time during storage was caused by bacteria and endogenous protein hydrolase. Songsaeng et al. [18] also suggested that the VBN value was approximately doubled by using both individual quick freezer (IQF) and contact metal plate freezer (CPF) for oysters stored at −20 • C for 48 weeks.
It can be found from Table 1 that the VBN values show an upward trend with time under any circumstance. For IFOM, the lowest is IPG at 9.6 ± 0.2 and the highest is I-c at 14.3 ± 0.3, while in BFOM, BPG is the lowest at 9.1 ± 0.2 and B-c has the highest at 13.6 ± 0.3. Additionally, it was not until about the 24th week that the changes in VBN, recorded from all subgroups, started to become more obvious. Notably, in Table 1, the result shows that there is a significant difference between IG and IPG; which is because of the addition of polyphosphate to the latter, i.e., IPG has a lower VBN value. In short, under the same condition of applying glazing, by employing the treatment of immersing oyster meats into polyphosphate, the effect of freshness on preserving is better. However, for IP (without ice-glazing) and IPG (with ice-glazing), the result shows no significant difference between the two; that is, in the state of immersing polyphosphate, even if we incorporate the ice-glazing treatment, there is only a very small effect. In other words, if frozen oysters are immersed in polyphosphate, the fresh-keeping effect is greater and stronger than that of glazed ones. Furthermore, the fresh-keeping effect of adding ice-glazing treatment is not relatively significant. In addition, a similar result can be gained for BFOM.
To sum up, immersing polyphosphate plays a core role in maintaining the quality of oyster meat, no matter whether ice-glazing does its job or not, for both IFOM and BFOM; that is, BPG and IPG are the best performers and have the most impact on the quality of oyster meat, while IP and BP come in second.

Color Attributes of Oyster during Frozen Storage
During frozen storage, the surface color of food might change due to the autoxidation of fats. More likely, the sublimation of the internal ice crystals would produce tiny holes that result in fat being oxidized inside the food. The former phenomenon is called rancidification, and the latter phenomenon is freezer-burn. After oil autoxidation and freeze burn occur, the color of food will become yellowish to dark brown [39]. Wasowicz et al. [40] reported that the oxidation of fat results in a color change in frozen food. The color becomes more and more yellowish and dark brown with the occurrence of oxidized fat, and the intensity increases as the storage time increases. Therefore, the L*, a*, and b* values come in handy and serve as indicators for frozen oyster quality. Songsaeng et al. [18] also pointed out that the appearance and color of oysters tended to deteriorate remarkably after 48 weeks. Looking at Table 2, it can be found that the L* value of the individual frozen oyster decreased from about 74.0 at the beginning to 62-67 at the 48th week, and the IG group was the lowest, while the IPG group was the largest. The L* value of block frozen oysters also decreased to 60-67, and B-c and BG were the lowest, whilst BPG was the largest. Among them, individually frozen oysters began to show differences at the 36th week, while block frozen oysters began to reveal differences at the 24th week. Table 3 shows that, for both IFOM and BFOM, the a* value has turned positive since week 12. This result indicates that the oyster color has changed from greenish to reddish, presuming it was induced by the oxidation of fat during the storage, which was caused by either rancidification or freezer burn, including the loss of water, the oxidation of fat and protein [41]. However, it can be found from Table 4 that the b* value gradually increases as the freezing time increases, which means the more time the oyster meats are frozen, the darker yellow they become. Wasowicz et al. [40] ascertained that the change in color was in accordance with the occurrence of oxidation during the storage period, making it appear to be more yellowish and darker brown. Apparently, the L*, a*, and b* values successfully act as a quality index for frozen oysters.

Total Aerobic Plate Count, TAPC
The total aerobic plate counts of frozen oysters, shown in Table 5, during storage of 48 weeks, were in the range of 10 3 to 10 4 CFU/g. A similar result was reported by Songsaeng et al. [18], stating the TAPC of frozen oysters was approximately 10 3 to 10 4 (CFU/g) during a year of frozen storage. The TAPC decreased significantly from 10 4 to 10 3 in the IPG and BP groups at week 48, after week 24 in the IP group, and after week 36 in the BPG group. Aside from that, little has changed, during the low temperature of −25 • C. The main reason for that was the confined environment of low temperatures, which caused microorganisms to go into a hibernating state.

Drip Loss
No matter how you handle frozen livestock or aquatic products, drips from thawing will always be there. It is known from the reports of many references that the amount of drip loss is closely related to the means of pre-freezing. Dripping not only reduces the weight and loses the nutrients of food, but also creates a better place and source for microorganisms to grow. Schwartz et al. [27] and Thorarinsdottir et al. [42] indicated that after phosphate treatment, it will increase the weight (increase the water holding capacity). Balasundari et al. [43] stated that the drip loss of oysters reached 19.8% after being frozen for 150 days. Boonsumrej et al. [25] explained that a large amount of water loss was found when tiger prawn (Penaeus monodor) was freezing, the same happened when thawing (drip loss), both of which should be combined and treated as drip loss. Songsaeng et al. [18] said that once individual frozen oysters were frozen at −20 • C for 48 weeks, the drip loss reached 16.6-27.5%; speculating that the cell and tissue structure suffering damage was due to the growth of ice crystals. Kaale et al. [44] proposed to increase the water holding capacity of frozen food and prevent drip loss.
In conclusion, whether to use ice-glazing (between I-c and IG, B-c and BG groups) or whether to add polyphosphate will affect the amount of drip loss. And there are significant differences between I-c and IP, B-c and BP, IG and IPG, BG and BPG. It can be seen from the experimental results that in terms of drip loss, there are extremely significant differences between IPG and I-c, as well as between BPG and B-c. Secondly, there are significant differences between IP and I-c, and between BP and B-c. In addition, the pH changes of IFOM and BFOM during storage ranged from 6.5 ± 0.2 to 6.9 ± 0.2, with only a small difference between the two.

Water Holding Capacity
Wang et al. [8] referred to the WHC of tuna steaks, with and without ice-glazing, dropping to 65.81% and 51.36%, respectively. Pinyosak et al. [45] also emphasized that when shrimp were stored at −18 • C for 24 h, WHC% decreased by about 4.5%. From Figure 1a, the WHC% of IFOM will decrease with the increase of freezing time, in which I-c declined the most. On the contrary, IPG is the winner with a high WHC%. On top of that, a huge difference developed between I-c and IPG. Within related pair groups, no significant differences existed between each other: I-c and IG and IP and IPG. From this plot, we find that oyster meat with polyphosphate care, achieved a higher level of WHC% than those with ice-glazing care. Figure 1b recognized the outcome by displaying an identical result from both IFOM and BFOM. In particular, BFOM seemed to be a little bit higher in WHC% than those of IFOM, but there was no significant difference in comparison with IFOM.

Effects of Repeated Freezing-Thawing on the Quality of Block Quick Freezing Oysters
When consumers buy frozen oysters, if there are too many frozen oysters (such as block frozen oysters), they must thaw them all before cooking, then take out an appropriate number of raw oysters, and then re-freeze the remaining oysters, and repeat this process until they are used up. In this case, the quality of the oysters should change significantly. For individually frozen oysters, the desired amount can be removed directly for the cooking process without repeated freezing and thawing. It can be seen from Table 8 that after 4 times of repeated freezing-thawing, the drip loss% gradually increased from the initial average of 10.1% to 33.6-40.1% in the 4th time, and the WHC% also decreased from an average of 88.3% to 58.1-63.2%; the score of cooked taste also dropped from the initial 8.5-9.0 to 5.5-6.5. Whether there is repeated freezing-thawing, the number of repetitions has a great impact on the quality of block frozen oysters, which also shows that the processing type of block frozen oysters is not good for consumers.

Effects of Repeated Freezing-Thawing on the Quality of Block Quick Freezing Oysters
When consumers buy frozen oysters, if there are too many frozen oysters (such as block frozen oysters), they must thaw them all before cooking, then take out an appropriate number of raw oysters, and then re-freeze the remaining oysters, and repeat this process until they are used up. In this case, the quality of the oysters should change significantly. For individually frozen oysters, the desired amount can be removed directly for the cooking process without repeated freezing and thawing. It can be seen from Table 8 that after 4 times of repeated freezing-thawing, the drip loss% gradually increased from the initial average of 10.1% to 33.6-40.1% in the 4th time, and the WHC% also decreased from an average of 88.3% to 58.1-63.2%; the score of cooked taste also dropped from the initial 8.5-9.0 to 5.5-6.5. Whether there is repeated freezing-thawing, the number of repetitions has a great impact on the quality of block frozen oysters, which also shows that the processing type of block frozen oysters is not good for consumers. Values are mean ± SD of 3 replicates. abcde Mean values in the same row followed by different letters are significantly different (p < 0.05). ABCD Mean values in the same column followed by different letters are significantly different (p < 0.05).

Conclusions
In terms of VBN, a recorded high trend is shown in all sampled groups as time increases during freezing storage. I-c and B-c, both without ice-glazing, are the highest values with the biggest difference from the others. Instead, IPG and BPG perform the best at the lowest values without significant differences from each other. In a word, the quantity has nothing to do with VBN, but the treatments-impregnated polyphosphate and ice-glazing-do. Besides, IPG and BPG work best with regard to water holding capacity (WHC).
Further analysis of the correlation between VBN and WHC% has shown that VBN itself stands for the degree of protein degradation by microorganisms or enzymes, but WHC% is closely related to the protein content, which means that high VBN-worse freshness-leads to lower WHC%.
For drip loss, the IPG and BPG are the best, and there is also no significant difference between them as well. In terms of appearance and color, as the freeze time increases, the lower the L* value, the lower or darker the brightness. The trend of the a* value is similar to the L* value. As the freezing time increases, it changes from a negative value to a positive value, and the value becomes higher and higher, which means that the color changes from green to red. On the other hand, the b* value showed the opposite trend. As the freezing time increased, the measured value increased as well, the more yellow or tan color appeared. These changes should be related to drip loss, protein degradation, lipid oxidation, and other factors. In terms of quality, it should be construed as having low freshness.
BPG and IPG, being polyphosphate impregnated and ice-glazed, have been verified to be the most effective and best in the way of preserving oyster meat, without any significant difference in between. If frozen oysters are to reduce the amount of drip loss or increase the water holding capacity during storage, the effect of adding phosphate should be better than ice coating. In addition, in terms of repeated freezing-thawing, the higher the number of repetitions, the greater the impact on the quality of frozen oysters. This can be attributed to the fact that too much drip loss and reduced water holding capacity result in poor size, shape, and taste after cooking, which is also the main reason why the block frozen oysters are not highly rated by consumers.