Effect of Oryctolagus cuniculus (rabbit) rennet on the texture, rheology, and sensory properties of white cheese

Abstract Calf rennet has long been used in cheese‐making. Because of calf rennet shortage and high cost, novel proteases were needed to meet industry's increasing enzyme demand. Recombinant chymosins and camel chymosin were started to be used in the industry. There is no study in the literature subjecting use of rabbit rennet in cheese production. Chemical, rheological, and sensorial characteristics of white cheese made with rabbit rennet were investigated in this study. Quality characteristics of rabbit rennet cheese (RC) were compared to cheeses produced with commercial calf (CC) and camel chymosins (CLC). RC and CLC exhibited higher hardness and dynamic moduli values throughout the storage as compared to CC. Although moisture levels of cheese samples were similar at day 60, CC had much lower hardness and dynamic moduli values than CLC and RC. While the appearance and structure were better for CLC, the highest odor and taste scores were obtained by RC during 60 days of storage. The results of this investigation proposed that rabbit rennet could be a suitable milk coagulant for white cheese production. Our results showed that rabbit rennet has comparable cheese‐making performance with camel chymosin and could be a good alternative for calf chymosin.

Many natural proteinases have the potential to coagulate milk and form visible curd. However, the number of proteases that could be employed in cheese-making is very limited. Chymosin, an aspartic proteinase, coagulates milk by specifically breaking the bonds established between amino acids present in the casein structure.
Therefore, novel milk-coagulating agents with a high specificity at the Phe(105)-Met(106) peptide bond and limited proteolytic activity are highly desirable by the industry. Good-quality cheese production is related to high milk-clotting activity and specificity of the enzyme on κ-casein Phe(105)-Met(106) bond (Elagamy, 2000;Fox, 1969).
Enzymes with low specificity may destroy other milk proteins and peptide bonds causing yield losses, formation of off-flavors, excessive softening, and other functional defects. Excessive proteolysis during storage limits the shelf-life of the cheese. For that reason, cheese industry is looking for enzymes with low proteolytic activity.
Proteolysis results in formation of bitter peptides. These peptides are more dominant and tangible in reduced fat and salt cheeses, and use of chymosin with low proteolytic activity rather than the traditional calf rennet is required for those productions (Govindasamy- Lucey, Lu, Jaeggi, Johnson, & Lucey, 2010).
Nowadays, scientists are still in pursuit of new coagulant sources for cheese production. Rabbit rennet has long traditionally been used in manufacturing of highly preferred cheeses in south-east region of Anatolia. This type of cheese is demanded and consumed admiringly due to its superior taste and properties in storage period such as low bitterness and softening. It is suggested that rabbit abomasum could be a good source of rennet. However, this enzyme has not been investigated extensively for commercial exploitation (Rao & Dutta, 1981). This situation still maintains its continuity. The aim of this study was to evaluate the suitability of rabbit rennet in cheese production. For this purpose, three types of coagulants as rabbit rennet, calf chymosin, and camel chymosin were employed for the manufacture of white cheese. The cheese samples were assayed based on their chemical, rheological, and sensorial properties during 60 days of storage.

| Coagulants
The coagulants used in this research were calf chymosin (CHY-MAX TM M, 600 international milk-clotting units (IMCU) per ml; Chr-Hansen A/S, Hoersholm, Denmark), camel chymosin (CHY-MAX ™ M, 1000 IMCU/ml Chr-Hansen A/S), and rabbit rennet extracted from young rabbit stomach. Rabbit stomach was obtained from local market, Mardin, Derik, which was cleaned, salted, and dried in a ventilated area prior to use in experiments and stored properly.
Rabbit rennet extraction was carried out according to the method described by Lambert (1988). Briefly, 10 g of dried stomach tissue was soaked into 12% salt solution (1/10) (w/v) at room temperature and stirred on a magnetic stirrer for 5 min. After adjustment of pH of the mixture to 4.3 with 1 mol/L HCl, the solution was incubated at 35°C for 72 hr. The extract was filtered, and pH of the mixture was re-adjusted to 5.6 with NaOH and stored at 4°C until cheese production.

| Milk-clotting activity determination
Milk-clotting activities of enzymes were calculated according to Equation (1) to determine the amount of rabbit rennet, camel, and calf chymosin necessary to give a coagulum at desired cutting time.
One milk-clotting activity unit (MCA) was defined as the amount of enzyme required to clot 1 ml of substrate in 40 min at 35°C. The MCA was calculated using Equation (1): where "t" is the time (s) necessary for clot formation, "S" is the milk volume, and "E" is the enzyme volume.

| Cheese production
Three different types of coagulant were used in cheese production as rabbit rennet, commercial calf chymosin, and commercial camel chymosin. According to our preliminary tests, adding calf chymosin at the level of 600 IMCU/ml (0.6 ml diluted with 10 ml water) gave a coagulum with similar gel strength as camel chymosin at the level of 1,000 IMCU/ml (0.65 ml diluted with 10 ml water) and rabbit rennet extract (10.65 ml) at 60, 120, 120 min, respectively, for 45 L of milk. Cheese manufacturing carried out according to the method described by Hayaloglu, Guvena, and Fox (2002) with some modifications at Harran University Food Engineering Department laboratories. Prior to cheese production, milk (3% fat) was pasteurized at 63°C for 30 min., cooled down to 35°C temperature, pH was fixed to 5.9 using citric acid, and CaCl 2 (0.02% w/v) was added. To obtain coagulum at desired cutting time (60 min at 35°C), rabbit rennet (10.65 ml), calf chymosin (0.6 ml), and camel chymosin (0.65 ml) were added to 45 L milk. After cutting the milk gel and draining the whey, curds were dry-salted (2.25 g/L milk), pressed, and cold-stored. Cheese samples were vacuum-packed the next day and stored at 4°C for 60 days. Cheese-making trial was performed in two replicates. Physicochemical, sensorial, rheological, and textural properties of the samples were evaluated at every 30 days.

| Analysis of composition
The composition of cheese samples (pH, moisture%, protein%, fat%, salt%) was assessed on the day of production and at every 30 days during 60 days of storage period according to the method described

| Sensory analysis
Cheese samples were stored for 60 days at 4°C. Sensory characteristics of stored cheeses were evaluated by panelists at 0, 15, 30, 60 days of storage. A panel composed of 10 members evaluated the odor, taste, structure, inner and outer appearance of cheese with a 5-point product-specific descriptive intensity scale for each textural attribute. In this evaluation, 1 point represents "low" and 5 points represent "high" quality of cheese for each parameter examined. Flavor properties were defined according to Drake, McIngvale, Cadwallader, and Civille (2001) and Drake et al. (2005) and texture characteristics were evaluated according to Brown, Foegeding, Daubert, Drake, and Gompertz (2003).

| Measurement of proteolysis
Proteolysis of the cheese samples was assessed by urea polyacrylamide gel electrophoresis (Urea-PAGE) (12.5% total acrylamide, 4% cross-linking agent, pH 8.9) according to the method of Andrews (1983). Urea-PAGE gels were monitored using Bio-imaging systems (mini BIS PRO, Israel) and photographed.

| Measurement of the rheological and textural properties
The rheological properties of the cheeses were studied with a rheometer (Kinexus Pro+, Malvern Inst., Worcestershire, UK) using SAOS temperature sweep test as described by Govindasamy-Lucey, Jaeggi, Johnson, Wang, and Lucey (2005) with small modifications.
Cheese samples were cut into disks at 3 mm height and 20 mm diameter. Storage modulus (G′), loss modulus (G″), and loss tangent (LT) values were measured while heating the cheeses from 10 to 80°C at 1°C/30 s. A 20-mm parallel plate was used, and the cheese was subjected to a strain of 0.5% at a frequency of 0.08 Hz. Texture analysis was performed using a Texture Analyzer TA-XT2 (Stable Micro Systems, Godalming, Surrey, UK). Cheese samples were cut into 2 × 2 × 2 cm cubes. Texture profile analysis (TPA) was performed at 25% strain; texture parameters were calculated as previously described by Bourne (1978).

| Statistical analysis
SPSS version 16 (SPSS Inc., Chicago, IL, USA) was used for analyzing the data obtained in this study. Analysis of variance (ANOVA) was employed to establish statistical differences between the physicochemical parameter values, texture profile analysis results, and sensory analysis scores according to rennet type, ripening time, and the interaction between those two factors.

| Cheese composition and yield
Composition, yield, and pH values of the cheese samples are given in be due to lipolysis and formation of free fatty acids. Cheese yield of the coagulants is shown in Table 1. The maximum yield was obtained in CC which was associated with its higher moisture content, and moisture-adjusted yield levels were similar for CC, RC, and CLC with no significant difference (p < .05). Even though the difference is not significant, it should be noted that RC and CLC moistureadjusted yield values were numerically higher than that of CC.
In a study of Cheddar cheese produced by camel and calf chymosin, although yield did not differ significantly, moisture-adjusted yield for camel chymosin was numerically higher than that of calf chymosin as observed in our study (Bansal et al., 2009). At the beginning of the storage period, CC samples had the highest moisture content in comparison with CLC and RC (Figure 1).

| Proteolysis of cheese
Water-insoluble fractions of cheese samples during storage are displayed in Figure 2. β-CN was hydrolyzed mainly to β-CN dation rates compared to other cheese types.

| Rheological and textural properties of white cheese
Storage and loss moduli values of RC cheese at day 1 were similar to CLC, while CC exhibited the lowest values (Table 2). Dynamic moduli values indicate the total number and strength of the bonds in cheese matrix (Lucey, Johnson, & Horne, 2003 Figure 3).
Texture profile analysis results of cheese samples are given in Table 3. CC had the lowest hardness at day 1, while RC and CLC were similar. CLC exhibited higher hardness values throughout the storage, while CC showed the lowest values. In previous studies using camel chymosin, it was also demonstrated that cheeses made with camel chymosin had higher hardness at storage which was contributed to its low proteolytic activity (Bansal et al., 2009;Govindasamy-Lucey et al., 2010;Grant, 2011;Kappeler et al., 2006

| Sensorial analysis
Sensory evaluation is important for determining the eating quality of cheese and its consumer acceptability (Delahunty & Drake, 2004). From past to present, many of coagulant sources were tried out in cheese-making but only some of them were accepted due to sensorial characteristics of produced cheeses. The sensory analyses results of CC, RC, and CLC samples are given in Table 4.
At the beginning of the storage; taste, appearance, and odor of all three cheese samples were similar, while structure of CC was found inferior to RC and CLC. There were no significant differences between the appearance of RC and CLC (p > .05) during  Values are means ± standard deviations. a,b Means within a row with different superscript letters are significantly different (LSD, p < .05). A,B,C,D Means within a column with different superscript letters are significantly different (LSD, p < .05).