Effect of Abalone Hydrolysates Encapsulated by Double Emulsion on the Physicochemical and Sensorial Properties of Fresh Cheese

Commercial pasteurized milk was purchased from the Seoul Dairy Co-op (Korea). Liquid animal rennet, edible citric acid, and calcium chloride used for the production of cheese curd were obtained from Cheesemaking Supply Co. (USA), Jungbunzlauer (Switzerland), and Daejung Chemical Co. (Korea). To prepare the double emulsions, whey protein isolate (WPI) (Milk Specialties Global, USA), edible medium-chain triglyceride oil (MCT oil) (Bolak Co., Ltd., Korea), polyglycerol polyricinoleate (PGPR) (Il-sin Co., Ltd., Korea), k-carrageenan (MSC Co., Ltd., Korea), polyoxyethylene sorbitan monooleate (Tween® 80) (Shamchun Pure Chemical Co., Ltd., Korea), abalone hydrolysate (Woosung F&B Co., Ltd, Korea), and salt (Chungjungone, Deasang Co., Korea) were used. To observe the double emulsion, methylene blue trihydrate (Samchun pure chemicals Co., Ltd, Korea) and Oil Red O (Sigma-Aldrich, USA) was used as a dye.

The double emulsion was prepared by modified method of Gu et al. (2005). Table 1 shows the components of the double emulsion used for cheese production. The inner aqueous phase (W₁) was prepared by hydrating WPI (2%, w/v) in water. The oil phase (O) was MCT oil containing 8%, w/v, of PGPR as a surfactant for W₁/O emulsion. The external water phase (W₂) was prepared by dissolving k-carrageenan 1% (w/v) and Tween 80 (polyoxyethylene sorbitan monooleate) 2% (w/v) in 1% NaCl solution without or with abalone hydrolysate. The water in oil (W₁/O) emulsion was created by mixing the inner aqueous phase (W₁) and the oil phase (O) at a 1:4 ratio, followed by homogenization at 10,000 rpm for 2 min using a high-speed homogenizer (IKAT25, Digital ULTRA-TURRAX^®, Germany). The W₁/O emulsion was dispersed into the outer aqueous phase at a ratio of 1:4, and the mixture was homogenized at 5,000 rpm for 2 min using a high-speed homogenizer (IKAT25, Digital ULTRA-TURRAX^®, Germany).

The fresh cheese was prepared using a modified version of the method described by Lobato-Calleros et al. (2008) and Lobato-Calleros et al. (2007). Milk was stored at 5°C until use. Before mixing with the double emulsion, 500 g of milk was heated at 37°C in a shaking water bath (BF-30SB, Biofree, Korea) for 30 min, 60 rpm. The heated milk was added to 2 g of edible citric acid (0.4%, w/w), 0.5 mL of a 40% calcium chloride solution (0.1%, w/w), and liquid animal rennet (0.1%, v/w) for production of cheese curd. For firm coagulation, the water bath temperature was raised to 50°C for 50 min. After 50 min, 5 mL of 1% NaCl solution or one of four different double emulsions (W₁/O/W₂) was added. After a coagulation time of approximately 30 min, the curd was cut into 1 cm³ cubes, and then approximately 80% of the whey was drained. The curds were transferred to a cotton cloth in a sieve, stored at room temperature for 2 h, and the whey was removed. Then, the curds were placed in a plastic barrel, and stored in a refrigerator (4°C) overnight.

Freshly prepared double emulsions were observed at 400× magnification under an optical microscope (CX31, Olympus, Japan) coupled with a CCD camera (3.0 M, Olympus, Japan). To distinguish the internal water phase in the oil droplets, the water (W₁) in the W₁/O/W₂ double emulsions was stained with 0.5% methylene blue solution hydrated with distilled water, or the MCT oil was stained with Oil Red O. The 0.5% methylene blue solution was added to 1% of internal water phase volume, and Oil Red O was added to 1% of MCT oil volume. In order to distinguish the shape of double emulsion, the methylene blue and red oil O was used to dye internal water and oil phase, respectively, before homogenization.

To determine the yield of curd production, the weight of the milk used to make the cheese and that of the final cheese with all whey removed were measured. The yield was expressed as a percentage, as shown in the following equation.

Studies have shown that pH value affects cheese quality (Marchesseau et al., 1997). A 3 g sample of fresh cheese and 27 mL of distilled water were ground in a household blender (CNHR 26, Bosch, Slovenia) for 1 min. The pH values of samples were determined using a pH meter (Orion 3-STAR, Thermo Scientific, USA).

The moisture content was determined in triplicate using the method described by the AOAC (1990). Curd was dried by heating to extract the moisture. Two-gram samples of curd were put in weighing bottles and dried in an oven (WOF-105, Daihan Scientific, Korea) for 24 hours. Drying was continued until the samples showed no change in weight. The water content was determined by comparing the weight of the sample before and after drying.

Samples of fresh cheese were placed in 3-cm petri dishes and the color was determined using a color reader (CR-10, Konica Minolta Sensing Inc., Japan) calibrated with a white standard plate (L* = 94.51, a* = −0.66, b* = 3.31). The CIE L*, a*, and b* values were designated as indicators of lightness, redness, and yellowness, respectively. The total color difference (∆E) was numerically calculated by comparing the color of the controls and treated samples using the following equation.

TPA was performed using a texture analyzer (CT3, Brookfield Engineering Labs Inc., USA) equipped with a TA39 probe and a TA-RT-KI fixture. Before measuring, 20 g of sample was placed in a cylindrical vial (22 × 50 mm; width × height). Samples were penetrated to 50% of their original height at a test speed of 2 mm/s and a trigger load of 5 g. The analysis was carried out at least 6 times. Values for hardness (g_f), adhesiveness (mJ), cohesiveness, springiness (mm), and chewiness (mJ) were determined.

The salt content of the cheese samples was determined using a 2 g sample homogenized in 100 mL distilled water using a magnetic stirrer (MSH-20D, Daihan Scientific, Korea) at 300 rpm for 600 s. The salt content was determined with a salimeter (8603, Mettler Toledo GmbH, Switzerland).

After storage overnight in a refrigerator (4°C), a 1 g sample was cut from each of the four cheeses. Samples were coded with three-digit random numbers, and randomly assigned to 8 panelists trained to test flavor, salty intensity, oiliness, and overall preference. The panelists’ preference for each cheese was ranked on a hedonic scale (5 = like extremely, 4 = like moderately, 3 = moderately, 2 = dislike moderately, 1 = dislike extremely).

Fig. 1 clearly shows that the internal water phase was entrapped in the oil droplets in all samples. The image clearly shows the fine dispersion of the internal aqueous-phase droplets (W₁) inside the large oil droplets that were dispersed in the outer aqueous phase (W₂), confirming the formation of double emulsions. The double emulsions are classified by three types based on the size and number of internal aqueous phase droplets (Florence and Whiehill, 1981). Type A double emulsion is consisted of only one large internal droplet inside the oil phase droplet. For type B double emulsion, the internal aqueous phase is composed of several small droplets. Alternately, type C double emulsion has a large number of fine internal droplets. In our emulsion, the type C was dominant and many small droplets are encapsulated in the relatively larger droplets. This means that the emulsion is dominated by large droplets. Type C shows slower release of active substances than A or B (Iqbal et al., 2017).

Fig. 1. Optical microscopy images of the water in oil in water (W1/O/W2) emulsions at 400× magnification.

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The pH values of the fresh cheeses are given in Table 2. The pH of cheese did not change significantly with the type of emulsion. Oloffs et al. (1992) observed that desirable milk coagulation is directly correlated with the pH value of milk. The pH values of all samples ranged from 5.02 to 5.16, and the pH of samples treated with salt enhancers or double emulsions showed no significant differences as compared to the control (p>0.05).

Since the same amount of citric acid was added to all samples for pH control, the pH value did not differ significantly with double emulsion type. Table 2 presents the curd yield of the cheeses prepared using the various double emulsions. The curd yield value was <25% for all samples. The curd yield of samples C and D was higher than that of samples A and B indicating that addition of double emulsions containing abalone hydrolysate produces a higher curd yield than other double emulsions. In general, curd production is related to milk protein. Cow’s milk contains 3-5% protein, 20% of which is whey protein and 80% of which is casein (Patel et al., 2008). Ferragina et al. (2013) and Sturaro et al. (2015) reported that the mean curd yield was 15.04% and 20.86%, respectively. Lobato-Calleros et al. (2008) reported that there was a significant difference in curd yield when double emulsions containing gum arabic, carboxymethylcellulose, or low-methoxyl pectin were added to cheese.

Moisture contents of the fresh cheeses are shown in Table 2. The moisture contents of the fresh cheese ranged from 56.19% to 57.58% and there was no significant difference among samples (p>0.05). In contrast, Giroux et al. (2013) reported that the moisture content of cheese made with double emulsion was higher than controls. Cervantes et al. (1983) reported that increasing the salt content did not decrease the moisture content of cheese.

The averages of the L*, a*, and b* values of the cheese samples, and the ∆E values are shown in Table 3. For the control group, the average values for L*, a*, and b* were 88.31, −1.84, and 6.71, respectively. Sample A differed significantly between the treatment groups and the control group (p<0.05), owing to the addition of abalone hydrolysate which is a dark brown color. The b* value of sample B was lower than the others owing to the absence of abalone extract in the double emulsion. The color of food is an important sensory attribute that can change consumers’ flavor perception (Young et al., 2004). In the case of cheese, it is important to recognize that the color of the cheese could affect the attitude of the consumer (Singh, 2006). Evert-Arriagada et al. (2012) and Sant’Ana et al. (2013) studied the L*, a*, and b*values of fresh cheese were 93.54, −3.16, 16.34, and 95.32, −60.0, 9.45, respectively.

Kwak et al. (2010) observed that addition of anchovy soluble protein extract causes significant changes in the color value of sausage, and Famelart et al. (1999) reported that when hydrolysate is added to cheese instead of salt, the physicochemical properties of the cheese are changed.

The texture properties of the fresh cheese samples are presented in Table 4. Kaya (2002) observed that salt concentration and hardness are associated in cheese. However, here the control showed higher hardness and adhesiveness values than cheese treated with the double emulsions, in spite their having the same salt concentration (p<0.05). We hypothesized that the MCT oil in the double emulsion increased the tenderness of the cheese. Serrano et al. (2005) observed that adhesiveness and oiliness are associated with the presence of oil in cheese. Significant differences in cohesiveness were observed between the control and other cheeses (p<0.05). Lee et al. (2016) showed that addition of hydrolysate, such as abalone hydrolysate into cooked noodles did not significantly change the cohesiveness or chewiness, in contrast to the results reported here.

In order to compare the results of sensory test at next section, the concentration of salt was measured over time by using instrument. Fig. 2 showed salt release rate over time, which indicates the degree of salt concentration detected. Fig. 2A indicated that the value became constant after 600 s in order to observe the releasing ending point. Fig. 2B showed the magnified from the Fig. 2A from 0 s to 60 s for the food chewing time in mouth, generally. As shown in Fig. 2, relative to the values of other samples, the salt concentration of the cheese made with the double emulsion containing the salt in the inner or external phase (sample C, D) or without abalone hydrolysate (sample B) was highest at the beginning, indicating a high NaCl release rate. This indicates that the external water phase containing NaCl can be quickly released during stirring due to localization of the NaCl in the outer phase. Comparatively, the NaCl release rate in cheese made without the double emulsion was slower than with the double emulsion. At 30 s, the NaCl concentrations of sample D and the control were 0.62% and 0.34% (Fig. 2B). These results indicate that sample D had a saltier taste than the other treatments, in accordance with the results of the saltiness rankings from the sensory test (Table 5).

Fig. 2. Salt release rate of curd prepared using double emulsions.

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In general, the diffusion rate of electrolytes such as Na cations is driven by concentration gradients, due to differences in surfactants, electrolytes, osmotic regulators, and encapsulated compounds (Bonnet et al., 2010). In our system, the shear force applied during stirring can break up the cheese, which can induce release of the NaCl in the double emulsion to the external water phase. For approximately 10 min, the concentration of NaCl remained constant at 1.0%, except in sample B. In the samples made with the double emulsion containing an internal water phase, the NaCl concentration of the dispersed phase did not decrease over time compared to the control.

The results of the sensory test are shown in Table 5. The average values for salt intensity, oiliness, and overall preference in the control group were 2.13, 3.80, and 2.33, respectively. For the treatment group, except sample A, the average values of salt intensity, oiliness, and overall preference were 3.88, 2.80-3.20, and 3.07-3.80, respectively. The salt intensity and overall preference for the cheeses prepared with the double emulsions were significantly higher than those of the control or sample B (p<0.05). In particular, sample D showed the highest scores for salt intensity and overall preference. It was considered that the abalone hydrolysates can play a role as a salt enhancer in the W2 phase of the double emulsions in cheese unlike the other samples, therefore it was affected to the value of overall preference. Moreover, since the NaCl was quickly released from the external phase of the cheese, the saltiness of the cheese could be rapidly detected on the mouth, when the panel chewed the fresh cheese piece (Fig. 2). Generally, saltiness is associated with overall preference in food, and people tend to prefer food with a higher salt content (Matsuzuki et al., 2008). For oiliness values, the cheese made with the double emulsions differed significantly from the cheese made without the emulsions, perhaps due to the MCT oil in the emulsions. As mentioned above, this result may be associated with texture, shown in Table 2. Therefore, we propose that application of the double emulsion of cheese could promote the saltiness of cheese, which may lead to reduce of the salt in cheese production. Thus, we consider that double emulsions containing a salt enhancer can be used in preparation of cheese with a reduced salt concentration but the same taste.