Effects of free and encapsulated transglutaminase on the physicochemical, textural, microbial, sensorial, and microstructural properties of white cheese

Abstract In this study, the effect of free and encapsulated transglutaminase (TGase) on physicochemical, textural, microstructural, microbial, and sensorial properties of white cheese was investigated. For this purpose, different types of white cheese incorporated with 20 and 60 ppm free enzyme (F20 and F60) and encapsulated enzyme (E20 and E60) were prepared and then compared with control (C) white cheese without TGase. The results showed that the addition of encapsulated TGase significantly (p ˂ .05) increased protein and fat content, dry matter, nitrogen recovery, and pH, as well as the production yield of cheeses. The hardness of treated samples was increased during the storage time, while the reverse trend was observed for the control sample. F60 and E60 samples showed more oriented and compact structures compared with other samples. Based on the results of sensory evaluation, E60 sample received the highest taste and flavor scores. Generally, the physicochemical, sensorial, and microstructural properties of white chesses were improved by the presence of encapsulated enzyme in the formulation.

cheese manufacturing during years. Many attempts have been made by manufacturers to produce cheese with pleasant taste and aroma from liquid milk by different biochemical processing (Litopoulou-Tzanetaki, 2016;Reis & Malcata, 2011). The rapid growth of the world population has increased the real demand for developing various milk products such as cheese. Therefore, traditional cheese manufacturing has sought new production systems to increase yield production. For this purpose, different types of starter cultures, cheese production machines, as well as refrigeration and pasteurization systems were developed (Ghanimah, Hanafy, Hassanein, & Hashim, 2018;Johnson, 2017). The application of TGase is one of the most promising approaches to increase the production yield of dairy products.

| Enzyme encapsulation
Alginate microcapsules were prepared according to the method of Allan-Wojtas, Hansen, and Paulson (2008). Briefly, alginate powder (10 g/L) was dispersed in deionized distilled water (DDW) and kept 24 hr to completely dissolve the polymer. Then, Tgase was added at different concentrations (20,000 or 60,000 ppm) under stirring for 10 min. The prepared suspension was emulsified by adding into sunflower oil (100 g) containing 5 g/L Tween 80 and then stirred at 900 rpm for 15 min. To initiate the gelation process, 32 ml of an emulsion system containing 62.5 mM CaCl 2 , 60 g oil, and 5 g/L Tween 80 was added into the mixture and mixed for 15 min. Finally, 40 ml CaCl 2 (0.05 M) was added to form microcapsules. The prepared microcapsules were removed by filter paper.

| Cheesemaking
For making white cheese, at first, milk was pasteurized at 65°C for 5 min. Then, the pasteurized milk was cooled to 35°C and the starter culture and CaCl 2 were added at concentrations of 0.04 g/L kg and 0.1 g/L milk, respectively. After 30 min of incubation at 35°C, rennet (0.025 g/kg) and TGase (20-60 ppm) were added. For two experimental cheeses, TGase was directly used in the free form (F20 and F60) and the other two samples were formulated with encapsulated Tgase in alginate microcapsules. For the coagulation of milk, samples were incubated for 45 min 35°C. Coagulation was started after 10 min, and milk was gelled after 45 min. The prepared samples were cut in 1 cm 3 cubes and then kept constant for 20 min. Then, they were transferred into a cloth and pressed at 25°C for 2.5 hr.
Afterward, cheese samples were removed and cut to 4 × 6 × 6 cm 3 cubes. The cheese cubes were placed in sodium chloride solution (13% w/w) for 19 hr at 23°C. After the ripening process for 60 days at 5-6°C, different properties of white cheese samples were investigated (Hayaloglu, Guven, & Fox, 2002). The control cheeses were produced in similar ways without TGase addition.

| Chemical composition
Fat content, dry matter, protein content, and salt content were determined according to AOAC (Chemists & Horwitz, 1990). Nitrogen recovery from cheese was determined according to the method of Johnson, Chen, and Jaeggi (2001).

| The yield of cheese production
To obtain the yield of cheese production, the initial weight of prepared cheese (after 1 day of storage at room temperature) was di-

| Textural analysis
Textural analysis of samples was performed by double compression test (TPA) using Texture Analyzer (CT34500) to obtain different parameters including hardness, springiness, cohesiveness, gumminess, adhesiveness, and chewiness. Cheese samples were cut to cylinder shape with 15 mm diameter and 15 mm height and kept at 25°C for 0.5 hr. The compression height and test speed were 66.6% of the initial height and 0.5 mm/s, respectively.

| Scanning electron microscopy (SEM)
The microstructure of alginate microcapsules and cheese samples were observed with scanning electron microscopy (SEM) using the technique described by Rahimi, Khosrowshahi, Madadlou, and Aziznia (2007) with some modifications. A slice of chesses sample was separated and fixed by glutaraldehyde (2.5%, w/w) for 180 min. The prepared sample was washed by DDW for 6 min.
Ethanol (30%, 50%, 75%, and 99%) was used for dehydration of wet samples after washing with water for 30 min. Then, chloroform was used for the extraction of lipids (twice times, each time 15 min). The small pieces of dried and defatted samples were frozen by liquid nitrogen, then coated with a thin layer of gold for 15 min in a sputter-coater (Desk Sputter CoaterDSR1, Nanostructural Coating Co.). Sample images were taken by a scanning electron microscope (TESCAN vega3) operated at an accelerated voltage of 15.0 kV.

| Microbial count
Coliform, E. coli, yeast and mold, Salmonella and Staphylococcus were determined based on the ISIRI standard method.

| Sensorial analysis
Twenty panelists (Semi trained students of food science and technology department of Azad University of kazerun, 10 males and 10 females) in the range of 23-33 years old evaluated the sensorial attributes of cheese samples including taste, color, and odor (Pino et al., 2018). Samples were studied by a 5-point hedonic scale (1 = do not like, 5 = liked). Cheese samples were placed in airtight plastic containers and kept at 25°C for 120 min before analysis. To cleanse the palate, water was used by panelists. The sensorial evaluation was conducted every 20 days during 60 days storage period.

| Statistical analysis
Results were analyzed by SPSS V. 19.0.0. Significance differences between the mean values were determined by one-way ANOVA variance analysis. Duncan's multiple range test at p < .05 was used for comparison of means.

| Microstructure of microcapsules
The microstructure of microcapsules containing TGase was shown in Figure 1. Microcapsules were not spherical, and the size of Yield 7.30 ± 0.14d 8.50 ± 0.14c 9.05 ± 0.21a 8.40 ± 0.14c 8.75 ± 0.07b Note: Data represent mean ± standard deviation of three independent repeats. Different lowercase letters in each row indicate significant differences (p < .05).
of κ-casein during coagulation of milk protein (Karzan, Nawal, & Ashna, 2016). Similarly, Bönisch et al. (2008) reported that the increase of serum binding in gel structure increased the production yield, which was due to additional covalent bonds of casein in the gel structure.  (Aaltonen et al., 2014). Storage time had no significant effect on protein content. The results of fat content (Table 2) showed that the addition of 60 ppm TGase increased the fat content significantly; however, storage time had no significant effect. This may be related to the extra fat added to the cheese formulation due to the preparation of alginate capsules in an oil emulsification process (Kailasapathy & Lam, 2005). TGase can affect this parameter by changing the gel structure. Darnay, Králik, et al. (2017) and Darnay, Tóth, et al. (2017) also reported that by addition TGase to semi-hard cheese, fat content significantly (p ˂ .05) increased. In agreement with the obtained results for protein and fat content, the dry matter was increased as affected by TGase addition. Karzan et al. (2016) also reported that the modified cheese with TGase had a higher dry matter. The result of nitrogen recovery showed that the addition of enzyme had a significant effect on the nitrogen recovery and sample containing 60 ppm enzyme had the highest value. Johnson et al. (2001) reported the nitrogen recovery of their sample was around 74%. The results of salt content showed that samples containing encapsulated enzyme had the highest salt concentration, which is due to using of salts in preparing alginate microcapsule as carrier of TGase.

| pH
The pH of treated samples with free and encapsulated TGase during storage is reported in Table 3. The results showed that the con-

| Textural properties
Textural properties including hardness, springiness, cohesiveness, gumminess, adhesiveness, and chewiness of cheese samples treated with free and encapsulated TGase are shown in Table 4. In the control  Aaltonen et al. (2014) reported that samples treated with TGase had the most changes in their water content and hardness due to covalent bonds formed by TGase. Darnay, Králik, et al. (2017)) and Darnay, Tóth, et al. (2017) reported the same trend about the effect of TGase on the hardness parameter.
The results of springiness and cohesiveness showed that these parameters significantly increased during the storage time and samples containing 60 ppm of enzyme had the highest springiness and cohesiveness values. The results of adhesiveness showed that this parameter was significantly reduced during the storage time and samples containing 60 ppm of enzyme had the lowest adhesiveness. Ahmed et al. (2015) observed that TGase could improve the emulsion and foam-forming abilities, water-holding capacity, and viscosity in low fat cheese. García-Gómez et al. (2019) reported that the addition of TGase to cheese sample prepared with chymosin significantly enhanced springiness and chewiness by 9% and 19%, respectively. They also reported that cohesiveness of the sample treated with TGase was greater than control. These findings were similar to the results of other researchers (Özer, Hayaloglu, Yaman, Gürsoy, & Şener, 2013). F I G U R E 3 Scanning electron microscopy (SEM) of cheeses. (C) Control sample without Tgase, (F60) sample containing 60 ppm free enzyme, (F20) sample containing 20 ppm free enzyme, (E60) sample containing 60 ppm encapsulated enzyme, and (E20) sample containing 20 ppm encapsulated enzyme C F60 F20 E60 E20

| Microbial properties
Evaluation of microbial properties of samples showed that yeast and mold, coliform, E. coli, salmonella, and staphylococcus bacteria counts were in the permitted ISIRI standard range. The initial count of milk, processing temperature, processing time, and production condition can affect the microbial count of final products.
Temperature, production of acid lactic and H + , as well as the increase in dry matter and chloride content are the main factor in the viability of microorganism in the cheese (Caridi, Micari, Caparra, Cufari, & Sarullo, 2003).

| CON CLUS ION
This research is the first report regarding the effect of different concentrations of free and encapsulated TGase on techno-functional and textural properties of white cheese. The production yield, protein content, fat content, dry matter, nitrogen recovery, and pH of samples increased by addition of TGase. Textural properties were also improved after treating with TGase. Sensorial properties showed that samples prepared with 60 ppm of enzyme had the highest scores. However, sample containing 60 ppm of encapsulated enzyme received the highest acceptability. This study has represented that application of encapsulated TGase into cheese matrix is an effective tool to create acceptable texture and enhance production yield in cheese products.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
Production of this product was done in sterile condition and before consumption by panelist, microbial analysis was evaluated. Also sensorial evaluation in the supervision of Dr. Yazdanpanah in test panelist room was done.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.