Regulation and optimization of water activity and quality of intermediate-moisture potato frozen cake

ABSTRACT The deterioration of cakes during frozen storage is a notorious phenomenon. This study aimed to evaluate the effects of cryoprotectants (collagen peptide, sericin peptide, and curdlan) on the intermediate-moisture cake during frozen storage. The results showed that cryoprotectants had a positive impact on water state and properties of products. Compared with the control group, the intermediate-moisture cake with better water-holding capacity had better texture characteristics and flavor than the others had, including alleviating the decrement of hardness and chewiness and promoting the augment of sensory evaluation. This study provided more comprehensive theories for the effects of cryoprotectants on intermediate-moisture cake quality from the perspective of water state.


Introduction
Cakes are highly appreciated by consumers because of their good taste, beneficial nutritional profile and ready-to-eat convenience. However, great majority of cakes circulating in the market are not ready-to-sell, led to the development of methods for the production and preservation of bakery products such as freezing technology. Although this technique enables businesses to reduce production cost and time due to its advantages on centralized manufacturing and distribution process (Selomulyo & Zhou, 2007), the deterioration during frozen storage is a notorious phenomenon. For example, the moisture migration and redistribution causing localized softening and drying, and the formation and growth of ice crystals causing structural distortion (Cauvain, 1998;He & Hoseney, 1990). Thus, the amount of unfrozen water in matrix is a major cause of product degradation during frozen storage. To improve the stability of frozen cakes, the control of water content and the addition of improvers might overcome these problems.
Intermediate moisture foods (IMFs) generally refer to a group of foods with 10%-50% (w/w) moisture content and the water activity between 0.65 and 0.9 (Prabhakar, 2014). In general, IMFs are considered as microbiologically stable at room temperature and can maintain certain initial characteristics of fresh food products (N. Y. N. Y. Lu et al., 2016). Soft cakes, such as Parfait, usually have a short shelf life at room temperature. When its water activity is controlled at 0.687-0.810, its shelf life at room temperature will be prolonged to 3 months. If stored under refrigeration, the shelf life will be even longer (Jelen, 2000).
Although IMFs have a longer shelf life during frozen storage, the growth and recrystallization of ice caused by the repeated fluctuation of temperature will lead to a destruction of the structure, thus hindering the development of such products. In the food industry, adding cryoprotectants is one of the most effective methods to alleviate the quality deterioration of foods during frozen storage. Traditional commercial cryoprotectant, such as polyphosphates, sugars, alcohols, and their compounds, are no longer conform with the current consumers' pursuit of health.
Antifreeze proteins (AFPs) are peptides and glycopeptides that can depress freezing point and modify the morphology of ice crystals (Xu et al., 2016). They also can inhibit the recrystallization of ice crystals by binding to the surface of ice crystals through an adsorption-inhibition mechanism (Clarke et al., 2002). Studies have shown that collagen peptide can be used as a cryoprotectant to improve bread quality and affect the moisture migration of dough during frozen storage (Xu et al., 2016). Sericin peptide has the function of antifreezing protection which can prevent cells and tissues from freezing denaturation (Tsujimoto et al., 2001). Adding a certain amount of sericin peptide into frozen food can not only improve the nutritional value of the product, but also improve the texture characteristics of the product (Gong et al., 2019).
Hydrocolloids can compete for water with polymers like protein and starch, thereby controlling the migration of water and improving the stability of products during frozen storage (Selomulyo & Zhou, 2007). Curdlan, a kind of polysaccharide produced by microorganism fermentation, can be used as stabilizer, thickener, or texturizer in food. It also can be used as cryoprotectant due to its strong hydrophilicity and freeze-thawed stability (Cong et al., 2004). Liang et al. (Liang et al., 2021) reported that curdlan could improve the quality and gluten network of frozen-cooked noodles by preventing ice crystals from growth and recrystallization.
Therefore, this study evaluate the effects of cryoprotectants (collagen peptide, sericin peptide, curdlan) on the quality of intermediate-moisture cake during frozen storage. The effects of cryoprotectants on the water content, water activity, water mobility, textural characteristics, and sensory quality of cakes before and after frozen storage were systematically investigated. The aim of this study is to offer some new scientific insight for quality improvement of intermediate-moisture frozen cake during frozen storage, and expand the application potential of collagen peptide, sericin peptide, and curdlan in food industry.

Preparation of intermediate-moisture potato frozen cake
The cake was prepared with 225 g wheat flour, 42 g potato flour, 140 g sucrose, 10 g baking powder, 240 g egg, 100 g oil, 140 g honey, 1.125 g whey powder, 1.125 g monoglyceride, and 1.125 g thickening agent. Cakes with collagen peptide, sericin peptide, and curdlan contained 0.225 g, 1.125 g, or 2.25 g collagen peptide, sericin peptide, and curdlan (0.1%, 0.5%, and 1.0% wheat flour basis). The addition of three different cryoprotectants were optimized by using a three-level and three-factor orthogonal experiment (Table 1). Cake with no cryoprotectant was used as control. Then, the cake paste was mixed in a vertical mixer. The cake paste was immediately divided into 100 g and transferred to a paper tray, then baked at 190°C/180°C for 28 minutes. Cooling the baked cake to room temperature and stored at −18°C for 0, and 4 weeks.

Determination of water activity and water content
The methods of testing the water activity and water content as described by Shima et al. (Mehrabi et al., 2017) were used with a brief modification. Equilibration at room temperature for 2 hours, the water activity of the core part in the cake piece was measured at room temperature using an H-BD5m water activity tester. The water content of the samples before and after freezing was determined by the weight discrepancy after drying at 105°C.

Water mobility determination
The water mobility of cake samples was determined using LF-NMR Analyzer (Niumag Corporation, Shanghai, China) according to the method described by Li et al. (Li et al., 2014) with some modification. Fragments of 10 mm × 10 mm × 10 mm were sealed in PET/PE bags. The spin-spin relaxation time (T2) of samples was performed by running Carr-Purcell-Meiboom-Gill (CPMG) sequences. The measurement parameters were the proton resonance frequency of 24.5 MHz, the test period TW was 1500 ms, the cumulative sampling number was 5000, and RGI was 20.0.

Texture profile analysis (TPA)
Fragments of 40 × 20 × 20 mm were dissected from the center part of freeze-thawed cake. Texture parameters were measured using a texture analyzer equipped with a P36 probe (Stable Microsystems TA-XT2i, Scarsdale, NY, USA). The test parameters were set as follows: pre-test speed 2.0 mm/s, test speed 1.0 mm/s, post-test speed 4.0 mm/s, and compression degree at 20%. The measured parameters include hardness and chewiness (Karaoğlu et al., 2008).

Sensory evaluation
Six screened assessors rated the cake in terms of appearance, internal structure, hardness, viscosity, and flavor. The scoring criteria is shown in Table 2. Table 1. Formula optimization for the adding of three different cryoprotectants by using orthogonal experiment (L9 (43)).

Statistical analysis
All experiments were run in triplicate unless specified and the experimental data was expressed as means (standard deviations, SDs) of three independent experiments. All statistical analyses were performed by using SPSS 19.0 for variance analysis and Minitab 17.0 for range analysis. The significance level of P < .05 was used.

Regulating effect of cryoprotectants on the water activity and water content of potato frozen cake
As shown in Table 3, all the samples prepared based on the formula in Table 1 could meet the requirements of IMFs. The water activity of groups 1-9 was lower than those of the control group, while the water content was higher than those of the control group after 4 weeks frozen storage. The decrease of water activity and the increase of water content indicated that the water holding capacity of the cakes had been enhanced Kong et al. (Kong et al., 2016) reported that large ice crystals could cause physical damage to the food structure, which lead to the decrease of water holding capacity. They confirmed that AFPs could reduce the size of ice crystals and prevent the formation of large ice crystals, resulting a preservation of cellular structure. Wu et al. also reported that sericin peptides could interact with water molecules through hydrogen bonding, hydrophobic interactions, and non-bonding interactions, which could prevent water from forming ice (Wu et al., 2015). As reported by Nguyen et al., the structural match and hydrogen bond formation between ice surface and the collagen peptide could bind collagen peptide to ice, which could inhibit ice growth (Nguyen et al., 2018). Curdlan could form helical structure by the bonding of hydrogen bonds with water molecules, and the structure will not change during freezing and thawing, thus  The basic cake control formula with the adding of 0.5% basic additive was used as the control group.
T ij refers to the mean value of evaluation index for each level of one factor. R value refers to the range value.
Los distintos superíndices dentro de la misma columna indican diferencias significativas según la prueba PLSD de Fisher a un nivel de confianza de 0.05. Como grupo de control se utilizó la fórmula de control del pastel básico con la adición de 0.5% de aditivo básico.
Tij se refiere al valor medio del índice de evaluación para cada nivel de un factor.
El valor R se refiere al valor del rango.
curdlan could significantly improve the water-holding capacity of frozen products (Hatakeyama et al., 2016). From the R value by the range analysis (Table 3), it could be seen that after 4 weeks frozen storage, curdlan had the greatest impact on water activity, followed by collagen peptide, and sericin peptide had the smallest effect. Collagen peptide had the greatest impact on water content, followed by sericin peptide, and curdlan had the smallest effect.
It had been widely reported that moisture had an important influence on the delicate taste and bulky appearance of cakes. Generally, cakes with low water content had poor taste and appearance. However, high water content would lead to high water activity of the products, which affect the safety of the products. Since all the tested samples were intermediate-moisture food with water activities lower than 0.9, the higher water content of frozen cakes the better quality of them. From Table 3, we found group 7 had the highest water content, and the adding amount for the cryoprotectants of it was 1% collagen peptide, 0.1% sericin peptide, and 1% curdlan, respectively. This result was almost agreed with the above referred result that the high content adding of collagen peptide and curdlan would help to increase the water content, leading to a better water holding capacity.

Effect of cryoprotectants on the water mobility of potato frozen cake
The formation and recrystallization of ice crystals during frozen storage will destroy the network structure of cakes and cause the migration of water in them, which will reduce their water holding capacity (Matuda et al., 2008). Therefore, it is necessary to investigate the water mobility in frozen cakes for further understanding the effect of cryoprotectants on decreasing the quality deterioration of frozen cakes during frozen storage. Figure 1 was the T2 relaxation time distribution curve of a sample. The curve showed two peaks: T21 (1 ~ 10 ms), T22 (10 ~ 100 ms), which represented deep bound water and semi-bond water, respectively (Doona & Baik, 2007). The  deep bound water, which is closely combined with gluten, starch, etc. will not move during frozen storage, while the semibound water will migrate and form ice crystals during the frozen storage.
The peak area ratio of T21 and T22 were presented in Figure 2. Compared with the control group, the proportion of deep bound water in experimental groups was higher than that in the control group after 4 weeks frozen storage, indicating that the water holding capacity of potato frozen cakes were improved. Groups 6-8 had the highest proportion of deep bound water. Collagen peptide and sericin peptide could bind on ice surface to inhibit the formation and recrystallization of ice crystals, thereby reducing the damage of the gluten network of the cake and increasing the deep-bound water content of the system. This result was consistent with the results of water migration of pig skin antifreeze peptides reported by Xu et al. (Xu et al., 2016) and the antifreeze effect of sericin on dough reported by Gong et al. (Gong et al., 2019). Williams et al. had reported that curdlan could form irreversible gel at high temperatures to hold moisture in meat and flour products, and the gel formed can maintain stability under pH 2-12 and freeze-thaw conditions (Williams et al., 2011).
Combined with the above results of water activity and water content in Table 3, it could be inferred that the addition of collagen peptide and curdlan could reduce the migration of water in the intermediate-moisture potato frozen cake, thus increasing the water holding capacity and decreasing mechanical damage during frozen storage.

Effect of cryoprotectants on the texture characteristics of potato frozen cake
Results of TPA test for all potato frozen cakes were illustrated in Table 4. In this study, the hardness and chewiness of cake were used to evaluate the quality of potato frozen cakes. Table 4. Regulating effect of hardness and chewiness of potato frozen cake by the adding of different cryoprotectant.
Tabla 4. Efecto regulador de la dureza y la masticabilidad del pastel de papa congelado mediante la adición de diferentes crioprotectores. T ij refers to the mean value of evaluation index for each level of one factor. R j value refers to the range value.
Tij se refiere al valor medio del índice de evaluación para cada nivel de un factor.
El valor Rj se refiere al valor del rango. T ij refers to the mean value of evaluation index for each level of one factor. R j value refers to the range value.
Los distintos superíndices dentro de la misma columna indican una diferencia significativa según la prueba PLSD de Fisher a un nivel de confianza de 0.05.
Tij se refiere al valor medio del índice de evaluación para cada nivel de un factor.
El valor Rj se refiere al valor del rango.
Hardness is an important index for evaluating cake quality, which is closely associated with human perception of freshness. Chewiness refers to the time required to chew the cake to the suitable for swallowing (T. M. T. M. Lu et al., 2010). Therefore, high values of chewiness are related with dense, which is not desirable in cakes. As shown in Table 4, the hardness and chewiness of cakes gradually increased with frozen storage time. The increase in the hardness and chewiness as a consequence of frozen storage indicates the destruction of gluten network by ice crystals during the freezing process (Yadav et al., 2009). The hardness and chewiness of groups 3, 4, 5, 6, and 8 were significantly (P < .05) lower than those of the control group before freezing. After four weeks frozen storage, the hardness and chewiness of groups 3, 4, 6, 7, and 8 were lower than those of the control group. The improvement was similar to the phenomenon of water migration, which further proved that there was a certain correlation between water migration and product quality during frozen storage. Lahtinen et al. (Lahtinen et al., 1998) reported that water content is the most important factor affecting the softness of cakes. Mehmet et al. (Karaoglu et al., 2008) also reported a significant negative correlation between water content of cakes and cake firmness. Thus, the improvement of water holding capacity of cakes might be helpful to reduce its hardness and chewiness, and delay its quality deterioration during frozen storage.
Meanwhile, from the R value by the range analysis (Table 4), it could be seen that before freezing, collagen peptide had the largest impact on the hardness and chewiness of the cakes. After 4 weeks frozen storage, collagen peptide had the greatest impact on the chewiness. Thus, among the three cryoprotectants, collagen peptide had the greatest influence on the hardness and chewiness before and after frozen storage. This result was similar to the results of water activity and water content. High content of collagen peptides could improve the texture of cakes, which might be due to the inhibition of ice crystals (Nguyen et al., 2018). In addition, collagen peptides could also modify ice crystals to make them smaller and more evenly distributed, thus making the texture of cake delicate and soft (Ling et al., 2018).

Sensory evaluation of cake
According to the sensory evaluation scores in Table 5, after 4 weeks frozen storage, groups 7 and 8 had similar sensory evaluation scores with the control group. Meanwhile, as the R value shown in Table 6, it could be seen that after 4 weeks frozen storage, collagen peptide had the greatest impact on the sensory evaluation, followed by curdlan, and sericin peptide had the smallest effect. This result almost agreed with the above result that the high content addition of collagen peptide could help to increase the quality of cakes. Therefore, we could realize that the intermediate moisture cake with better water holding capacity would have better sensory quality after freezing.

Conclusion
This study investigated the effects of cryoprotectants in frozen intermediate moisture cake. Collagen peptide, sericin peptide and curdlan were added in frozen intermediate moisture cakes to weaken the quality deterioration of cakes during frozen storage. The underlying mechanism of cryoprotectants on quality regulation and optimization during frozen storage was elucidated from water state and properties. In addition, the texture and sensory properties were also explored to analyze the quality of frozen cakes. The results showed that the improvement of water holding capacity improved the quality of intermediate-moisture cakes during frozen storage, including alleviating the decrement of hardness and chewiness and promoting the augment of sensory evaluation. The results provided a theoretical basis and technical reference for revealing the effects of cryoprotectants on water mobility, texture and flavor of intermediate-moisture cake during frozen storage, and expand the application potential of collagen peptide, sericin peptide and curdlan in food industry.