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Interaction between rennet source and transglutaminase in white fresh cheese production: Effect on physicochemical and textural properties
LWT - Food Science and Technology 113 (2019) 108279
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Interaction between rennet source and transglutaminase in white fresh cheese production: Effect on physicochemical and textural properties
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Belén García-Gómeza, Mª Lourdes Vázquez-Odériza, Nieves Muñoz-Ferreirob, Mª Ángeles Romero-Rodrígueza, Manuel Vázqueza,∗ a b
Department of Analytical Chemistry, Faculty of Science, University of Santiago de Compostela, 27002, Lugo, Spain Modestya Research Group, Department of Statistics, Mathematical Analysis and Optimization, University of Santiago de Compostela, 27002, Lugo, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Texture TPA Yield Cheese Rennet Transglutaminase
The aim of this study was to determine the effect of the rennet source and the interaction of rennet with TG on physicochemical and texture properties of fresh cheese made with pasteurized cow milk. The rennets used were: animal, recombinant chymosin, vegetal or microbial rennet. Cheeses with and without addition of TG was obtained. The rennet source did not affect statistically the activity water and water content of the cheese. The highest yield was obtained using recombinant chymosin. The rennet source showed important effects on texture parameters. Microbial and vegetal rennets increased the adhesiveness of cheeses. Cheeses coagulated with animal rennet were the most affected by the TG treatment, observing a significant decrease in hardness (62.19%), chewiness (75.09%) and springiness (43.75%). The adhesiveness and cohesiveness values also increased significantly. An increase of 0.69% in the yield was achieved when the TG was combined with animal rennet. The TG addition combined with animal rennet can be an effective way to obtain cheese products with innovative textural properties and slightly improved yield.
1. Introduction The manufacture of cheese has been a common practice of mankind for thousands of years. It basically involves the conversion of liquid milk into solid curd, followed or not by the biochemical evolution of the curd into ripened cheese (Reis & Malcata, 2011). The traditional manufacture of a cheese could not satisfy the demand for cheese, but the development of refrigeration systems, the commercial availability of the starters and the use of pasteurized milk allowed a qualitative and quantitative leap in cheese production. The implementation of new technologies is necessary to optimize the production process and satisfy the consumer demand (Ghanimah, Hanafy, Hassanein, & Hashim, 2018; Johnson, 2017). In addition to be a widely consumed product due to its sensory properties, it is necessary to highlight the important role in human nutrition throughout the history of humankind. However, dairy products in general and cheese in particular have been associated recently with negative effects on health. The nutritional importance of the cheese was confirmed in a recent study where it was observed that during milk fermentation in cheese manufacture the phospholipid concentration suffer a great increasing enhancing the cheese nutritional
∗
value (Ferreiro & Rodríguez-Otero, 2018; Zhi et al., 2017, 2018). In addition, recent studies suggest that bioactive compounds such as functional lipids (CLA), vitamins, antimicrobial peptides, γ-aminobutyric acid and exopolysaccharides with a potential effect on disease prevention could be released by some lactic acid bacteria (SantiagoLópez et al., 2018). Many researches are being carried out to improve the cheeses from the nutritional point of view without altering the sensorial properties or the shelf life, such as the reduction of the sodium content more than 30% using salt replacers (Carmi & Benjamin, 2017). Most varieties of cheese are produced by enzymatic coagulation with rennet. Cheese has become a very important product in the dairy industry (Fox, Guinee, Cogan, & McSweeney, 2016; Pinheiro et al., 2012). Nowadays functional dairy products present in market are mainly made from cow milk although cheese manufactured with milk of small ruminants as sheep and goats are also frequent (Ardelean, Otto, Jaros, & Rohm, 2012; Llamas-arriba et al., 2019; Pinheiro et al., 2012). 1.0) Enzymatic coagulation of milk is a crucial step in cheesemaking process. Rennet is a mixture of proteolytic and lipolytic enzymes such as chymosin, pepsin and lipase. Its activity is required for enzymatic milk coagulation. Rennet is the responsible of the degradation of casein proteins through disruption of covalent peptide bonds of
Corresponding author. E-mail address: [email protected] (M. Vázquez).
https://doi.org/10.1016/j.lwt.2019.108279 Received 14 May 2019; Received in revised form 7 June 2019; Accepted 17 June 2019 Available online 21 June 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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κ-casein formed between phenylalanine and methionine (Selin et al., 2018). The enzymatic cleavage causes the destabilization of the casein micelles that are aggregated and a three-dimensional protein matrix is obtained forming the curd (Amira, Besbes, Attia, & Blecker, 2017; Fox et al., 2016). The main role of proteolysis is to be responsible of the curd formation. Moreover, it is related with the release of amino acids as precursors of catabolic reactions in which flavor compounds are released (Fox et al., 2016; Hayaloglu & Karabulut, 2013; Katsiari, Alichanidis, Voutsinas, & Roussis, 2000). 2.0) In cheese manufacturing, the coagulation of milk is traditionally made by calf rennet chymosin obtained from the abomasum of nursing animals. Rennet extracted from calf, kid or lamb abomasus were the primary commercial animal source. Other enzymes are capable of initiating the proteolysis like aspartic proteinases. They can be extracted from some plants, microorganism and different ruminants such as pig, lamb, goat and rabbit (Balabanova, Ivanova, & Vlaseva, 2017; Selin et al., 2018). Nowadays, more than 90% of the rennet used in the cheese is fermentation-produced chymosin. The use of substitutional enzymes of calf rennet can be an interesting alternative to meet the demand of lacto-vegetarians people and it has the advantage of being both kosher and halal approved (Jacob, Jaros, & Rohm, 2011; Johnson, 2017). 3.0) Rheological and sensory properties of the cheese are related to the enzymatic activity (Amira et al., 2017). The rennet substitutes must have a relatively weak proteolytic activity in order to maintain the quality of the product since an excessive proteolysis may cause yield losses during manufacture due to denaturing of к-casein in the whey and the development of bitter taste in the final product (Fox et al., 2016; Mazorra-Manzano et al., 2013). For this reason, chymosin is particularly suitable for the cheesemaking due to its low proteolytic activity and its high milk clotting activity. The most important substitutes that meet the requirements of cheese making include microbial, recombinant and plant-based enzymes (Shah & Mir, 2014). Plant proteases are classified into several groups according to the catalytic mechanism used during the hydrolytic process. Many enzymes used as milk coagulants contain aspartic proteases. Enzymes with proteolytic action such as cysteine and serine are also used (Shah & Mir, 2014). Aspartic proteases are extracted mainly from the flowers of many varieties of plant species (Amira et al., 2017). Rennet extracted from dried flowers of the wild thistle Cynara cardunculus has been employed on manufacture of traditional semi-hard cheeses from raw ovine milk at the farm level since ancient times in different Spanish regions (Almeida & Simões, 2018; Sousa & Malcata, 1997). Its application was also studied in cow's milk (Sousa & Malcata, 2002). The best option recommended is the use of cardoon extract formulations (Almeida & Simões, 2018). Many extracellular microbial proteases act similar like chymosin. The advantage of microbial rennet is their availability since it can be easily obtained by fermentation. However, the increased activity of those proteins during cheese making leads to an excessive degradation of proteins. These are lost in the serum and have a negative effect on the cheese yield (Jacob, Jaros, et al., 2011). The excessive proteolytic activity was controlled by chemical modifications and using genetic engineering tools on the microbial organism (Yegin & Dekker, 2013). Nowadays, microbial proteases are obtained from Rhizomucor miehei, Rhizomucor pusillus, and Cryphonectria parasitica (Balabanova et al., 2017; Sumantha, Larroche, & Pandey, 2006; Yegin & Dekker, 2013). They have been established for large-scale production being commercialized and applied in cheese manufacturing (Jacob, Nöbel, Jaros, & Rohm, 2011). The aspartic protease produced by Rhizomucor miehei is the most used in cheese manufacture and is commercially available at different levels of thermostability and purity (Jacob, Jaros, et al., 2011). Therefore, nowadays four sources of rennet are available in the market: calf rennet, recombinant chymosin, vegetal rennet or microbial rennet. To the best of our knowledge, there are not studies comparing
the effect of the four rennets on the same type of cheese. On the other hand, food industry is looking to develop new dairy products with novel physical and functional characteristics (Sharma, Lorenzen, & Qvist, 2001). In the cheese industry, it is also important to improve the yield. Enzymatic crosslinking with transglutaminase (TG) have a great potential to achieve both aims (Cozzolino et al., 2003; Gaspar & De Góes-Favoni, 2015; Kuraishi, Yamazaki, & Susa, 2001; Mahmood & Sebo, 2009; Martins et al., 2014; Taghi Gharibzahedi et al., 2018). TG can change the composition of proteins by crosslinking, amine incorporation and deamidation (García-Gómez, RomeroRodríguez, Vázquez-Odériz, Muñoz-Ferreiro, & Vázquez, 2018, 2019; Motoki & Seguro, 1998). Those reactions modify the functional properties of vegetable and animal proteins allowing to obtain products with improved rheological and sensory properties (Gauche, Vieira, Ogliari, & Bordignon-Luiz, 2008; Yokoyama, Nio, & Kikuchi, 2004). A microbial transglutaminase was isolated from Streptomyces mobaraensis at the late 1980s. It was Ca2+ independent and stable over a wide range of temperatures and pH (Ando et al., 1989). Furthermore, it was particularly useful for food industrial applications because it shows an improved reaction rate, broad substrate specificity for the acyl donor and low-cost mass production (Guerra-Rodríguez & Vázquez, 2014; Jaros, Partschefeld, Henle, & Rohm, 2006). The aim of this study was to determine the effect of the rennet source and the interaction of rennet with microbial transglutaminase on physicochemical and texture properties of fresh cheese made with pasteurized cow milk. 2. Materials and methods 2.1. Raw materials Commercially available pasteurized full fat cow's milk (protein, 3.1 g/100 g; fat, 3.6 g/100 g; ESM, 3.6 g/100 g. Four types of coagulants were used: animal rennet from calf, vegetal rennet from Cynara cardunculus, microbial rennet from Rhizomucor miehei and recombinant chymosin from Kluyveromyces lactis. All coagulants were supplied by Abiasa (Tui, Pontevedra, Spain). TG was produced in our laboratory following the manufacture process described in our Spanish patent ES-2376439 (Vázquez & Guerra-Rodriguez, 2012). A spectrophotometry procedure was used to measure the TG activity before. A calibration curve was made using Lglutamic acid γ-monohydroxamate (Sigma-Aldrich Corp, St. Louis, MO, USA). N-α-CBZ-gln-gly (Sigma-Aldrich Corp, St. Louis, MO, USA) was used as substrate. One unit of TG is defined as the formation of 1 micromol L-glutamic acid γ-monohydroxamate in 1 min at 37 °C (Grossowicz, Wainfan, Borek, & Waelsch, 1950). The measured activity previously to use were 470 U/g. 2.2. Cheese manufacturing The cheeses have been manufactured as described below. The first batch was coagulated with animal rennet (A); the second batch was coagulated with animal rennet and microbial transglutaminase (A + TG); the third batch was coagulated with recombinant chymosin (C); the fourth batch was coagulated with recombinant chymosin and microbial transglutaminase (C + TG), fifth batch was coagulated with vegetal rennet (V); the sixth batch was coagulated with vegetal rennet and microbial transglutaminase (V + TG); the seventh batch was coagulated with microbial rennet (M); and the eight batch was coagulated with microbial rennet and microbial transglutaminase (M + TG). The milk used to make the samples treated with TG (batch 2, 4, 6 and 8) was heated up to 40 °C, then, TG was added (0.8 U/g protein) and the mixture was incubated during 20 min at 38 °C before curd production. The milk used to make the batches without microbial transglutaminase (batch 1, 3, 5 and 7) was heated up to 38 °C. Then with the milk at 38 °C in all the batches, 0.25 mL/kg of coagulant, 2
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Fig. 1. Flow diagram for cheese production treated with transglutaminase (A) and without transglutaminase (B).
maximum height of the first compression. Adhesiveness was measured as the area under x-axis on compression graph curve. Springiness was calculated as the height of the product on the second compression divided by the height of the first peak. Cohesiveness was calculated as the ratio of the areas of the second and the first peak. Chewiness was calculated as hardness*cohesiveness*springiness (Castro-Briones et al., 2009). Eight samples were analyzed for each cheese trial.
0.3 mL/kg of CaCl2 (43.5%) and 6 g/kg of NaCl were added to obtain the curd. The curd obtained during the enzymatic coagulation was cut into cubes having size of 2 cm. The curd was separated from the whey and was placed in perforated cylindrical molds with 12 cm of diameter and 7 cm of height, without putting pressure. The curd was drained during 12 h. Then, cheeses were demolded and stored at 4 °C for at least 24 h before analyze them. Cheeses were sampled for instrumental texture analysis, physico-chemical analysis and yield measurement. The flow diagram of cheese manufacture and the analyses performed is shown in Fig. 1. Duplicate batches were performed.
2.4. Statistical analysis Two-way analysis of variance (ANOVA) was accomplished on the texture parameters considering the source of rennet (A, C, V and M), TG addition (yes/not) and their interaction as fixed sources of variation. It was considering the type of coagulant as treatment 1 with levels A, C, V and M and the TG addition as treatment 2 (yes/not). For the parameters Aw, W and Y, the Kruskal-Wallis test was applied when TG addition effect was analysed and Mann-Whitney U test was applied when the effect of the type of coagulant was studied (Granato, de Araújo Calado, & Jarvis, 2014). Principal Component Analysis (PCA) was applied in order to identify underlying factors (texture parameters, Aw, W and Y) that explain the pattern of correlations within the set of observed variables. The average for each sample for each variable were used to perform the data matrix for PCA. The variables have not the same units. Therefore they were scaled in order to give the same influence to each one (Granato et al., 2018; Le, Josse, & Husson, 2008a). Statistical calculations were performed using IBM SPSS Statistics 20 software for Windows (IBM, Armonk, NY, USA) for ANOVA and R software (R Core
2.3. Analysis Water content (W) was determined gravimetrically drying at 102 °C for 48 h by triplicate. Water activity (Aw) was measured using AquaLab meter (Pullman, WA, USA). W and Aw of fresh cheese were determined in the final product on triplicate samples. Yield (Y) of each batch was expressed as weight of cheese obtained from 100 g of milk employed for the production of cheese. Fresh cheese was cut into small cubes (2 × 2 × 1.5 cm). Texture profile was determined at room temperature using a TA-XTplus texturometer (Stable Micro System, Viena Court, UK). It was used a cylindrical aluminum probe (P/50) with 50-mm diameter. The compression speed was 60 mm/min. Samples were compressed to 60% of the original height. It was measured hardness, adhesiveness, springiness, cohesiveness and chewiness. Hardness was measured as the force at the 3
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Table 1 Results of water activity (Aw), water content and production yield for the cheeses with the rennet studied (no TG added). Aw
Water content (% w/w)
Yield (%)
63.79 63.60 68.02 68.91
17.78 20.19 19.83 16.57
Rennet source Animal Chymosin Vegetal Microbial
0.990 0.990 0.990 0.990
± ± ± ±
0.000 0.003 0.004 0.001
± ± ± ±
0.00 0.03 0.01 0.01
Team, 2018) using the FactoMineR package for the rest of statistical analysis (Le, Josse, & Husson, 2008b). Differences at a level of pvalue < 0.05 were considered significant. 3. Results 3.1. Effect of the rennet source Table 1 shows the results of Aw, water content and production yield. The Aw values did not change with the rennet source, showing values around 0.99. The samples showed similar water contents in cheeses with animal rennet and recombinant chymosin, but lower than those obtained with vegetal and microbial rennets. However, no significant differences were found (p-value > 0.05). Regarding the yield, the lowest yield was obtained using microbial rennet (16.6%) and the highest using recombinant chymosin (20.19%). The effect of the rennet source on cheese texture was determined by the TPA analysis. The mean values and standard deviations of the TPA parameters are given in Figs. 2 and 3. The cheeses obtained with animal rennet were significant harder than the others (p-value ≤0.05) (Fig. 2a). Chymosin and microbial rennet showed intermediate hardness (2672l.46 and 2594.43 g, respectively). The vegetal rennet produced cheeses that are significantly softer than the obtained with the other rennets (p-value ≤ 0.05). The cheeses obtained with animal rennet is a 40.17% harder than that obtained with vegetal rennet (2101.74 g). Cheeses coagulated with animal rennet showed the smallest adhesiveness (−49.96 g s). The highest adhesiveness was measured in the cheeses obtained with microbial rennet (−255.33 g s) (Fig. 2b). Microbial rennet gave cheeses 80.43% more adhesive than those obtained with animal rennet. Significant differences (p-value≤0.05) were found for all the rennets used, except for vegetal and microbial rennets. Microbial and vegetal rennets were similar between them and significantly different compared with chymosin and animal rennets (pvalue ≤ 0.05). Chewiness showed the same trend that hardness (Fig. 2c). The lowest values were observed in cheese coagulated with vegetal rennet and the highest using animal rennet. Cheese obtained with animal rennet showed the highest values for springiness, following by cheeses coagulated with chymosin and microbial rennet (Fig. 3a). Again, the vegetal rennet gave the lowest values. The differences observed in cohesiveness was smaller compared with the other texture parameters (Fig. 3b). The highest values were obtained using vegetal and microbial rennets. The lowest values were measured in samples coagulated with animal rennet and chymosin. Chymosin and animal rennet gave cheese with similar cohesiveness but significantly smaller (p-value≤0.05) than the obtained using vegetal and microbial rennet. The difference between microbial rennet and chymosin was 21.43%.
Fig. 2. Mean values and standard deviation for hardness, adhesiveness and chewiness. Different letters show significative statistical differences between the rennet source (A was coagulated with calf rennet; C was coagulated with recombinant chymosin; V was coagulated with vegetal rennet and M was coagulated with microbial rennet).
cheeses without TG, except in those coagulated with chymosin. The differences in Aw values were statistically significant for samples coagulated with chymosin and vegetable rennet. The addition of TG increased the water content of the samples coagulated with animal rennet and chymosin, 3.8 and 2.7% respectively. No statistically significant differences were found between the sample treated with TG and the untreated sample for any of the coagulants used. When TG was added, the differences observed in the yield were scarce, barely of the 0.5 and 0.7% for the animal and vegetable rennet, respectively. I was observed even a slight descent in the cheeses coagulated with chymosin and vegetable rennet. Fig. 4 show the TPA parameters obtained for the cheese coagulated with animal rennet. The cheeses coagulated with animal rennet with TG showed a decrease in hardness of 62.19%. Regards adhesiveness, the sample coagulated with animal rennet has also been affected by the TG addition (p-value≤0.05). Adhesiveness values decreased from −49.96 to −167.93 g s when TG was added. The chewiness value decreased by
3.2. Effect of the TG addition The four rennets have been studied separately to compare the effect of the transglutaminase addition in each of them (Table 2). The values of Aw were lower in the cheeses treated with TG compared with the 4
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Fig. 4. Mean values and standard deviation for the TPA parameters (hardness, adhesiveness, springiness, chewiness, springiness and cohesiveness) of cheese coagulated with animal rennet without TG (blue bars) and adding TG (orange bars). Different letters show significantly statistical differences between the sample treated with transglutaminase (TG) and the untreated one for the sample coagulated with calf rennet.
Fig. 3. Mean values and standard deviation for springiness and cohesiveness. Different letters show significative statistical differences between the rennet source (A was coagulated with calf rennet; C was coagulated with recombinant chymosin; V was coagulated with vegetal rennet and M was coagulated with microbial rennet). Table 2 Results of water activity (Aw), water content and yield for the samples with TG treatment. The asterisk shows significant statistical differences comparing rennet source when the transglutaminase was used. Rennet source
Aw
Animal Chymosin Vegetal Microbial
0.987 0.991 0.989 0.986
± 0.001 ± 0.002* ± 0.002* ± 0.002
Water content (% w/w)
Yield (%)
67.59 66.31 67.54 68.81
18.47 19.61 17.84 17.08
± ± ± ±
0.04 0.13 0.03 0.01
Fig. 5. Mean values and standard deviation for the TPA parameters (hardness, adhesiveness, springiness, chewiness, springiness and cohesiveness) of cheese coagulated with chymosin without TG (blue bars) and adding TG (orange bars). Different letters show significantly statistical differences between the sample treated with transglutaminase (TG) and the untreated one for the sample coagulated with recombinant chymosin.
75.09% when TG was added. The highest value for the springiness was that observed for the sample coagulated with animal rennet without TG. When TG has been added, springiness decreased by 43.75%. The TG addition were responsible of an increase in cohesiveness around 11.11% in the samples treated with animal rennet (A + TG) compared with the untreated sample (A). Fig. 5 show the TPA parameters obtained for the cheese coagulated with recombinant chymosin. Neither the hardness nor the adhesiveness of cheeses coagulated with recombinant chymosin have been affected by the addition of TG, obtained values around 2670 g and −120 g s, respectively. Opposing to what happened with animal rennet, the incorporation of TG in the manufacture of cheeses coagulated with recombinant chymosin significantly increases chewiness and springiness (Fig. 5). The observed increase was 19.19% for chewiness, 9.09% for springiness. The cohesiveness was also increased using TG like in the case of using animal rennet. Fig. 6 show the TPA parameters obtained for the cheese coagulated with vegetal rennet. Treatment with TG slightly increased all the texture parameters. The increase was lower than 10%, except for the cohesiveness that decreased by 16%. However, the differences were not
significantly. Fig. 7 show the TPA parameters obtained for the cheese coagulated with microbial rennet. The treatment with TG has been responsible for a decrease in all the texture parameters. The decrease in texture values were statistically significant for chewiness, springiness and cohesiveness. Chewiness was the parameter more affected by the addition of TG with a decrease of 21.18%. Finally, Principal Component Analysis was performed in order to facilitate the joint vision of the physicochemical variables, TPA parameters and the yield obtained for the different samples. The explained variance of the first and second factor accounts for 80.16%. Fig. 8 shows that the sample with a higher yield and Aw has been coagulated with recombinant chymosin without transglutaminase (C). The lowest yields and Aw have been observed in the samples coagulated with microbial rennet (M and M + TG). When the samples were treated with TG, the differences in yields comparing the different rennets were minimized. Comparing the rennet source, the sample coagulated with chymosin 5
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be due to the fact that the preincubation of the milk with the TG would have inhibited hydrolysis of k-casein during the coagulation, inhibiting the micelles aggregation. Consequently, the milk coagulation was affected (Bönisch, Heidebach, & Kulozik, 2008). Probably due to this fact, some researchers using chymosin as coagulant did not get the coagulation of the milk when incubated the milk with TG for 20 min at 20 °C before adding the coagulant. However, an increasing in cheese yield was observed when the transglutaminase was added after the clotting enzyme inducing the formation of the coagulum (Cozzolino et al., 2003). Similar results to those obtained by the latter researchers were observed in other study (Yüksel, Avci, & Erdem, 2011). However, milk coagulation was achieved by incubating with TG prior to adding the coagulant although an increase in renneting time was observed (Domagała et al., 2016). In other researches, the increase in yield achieved in TG treated white-brined cheese coagulated with animal rennet was attributed not only to a higher serum retention related with the alteration of the physical properties of the casein gels with a lower pore size but also a slight increase of protein fraction observed (Özer, Hayaloglu, Yaman, Gürsoy, & Şener, 2013). Our results show a strong correlation between water content and cohesiveness (Fig. 8). The sample coagulated with chymosin (C), it has been the least cohesive and with a lower water content. The cohesiveness values observed in the samples coagulated with animal rennet have been greater when transglutaminase has been added (Fig. 5). Those results were according with the findings of other researchers (Özer et al., 2013). The highest values of hardness, springiness and chewiness were observed in the sample coagulated with animal rennet. When this coagulant is combined with TG, a decrease in the values of those parameters was observed. Opposite our findings, other researchers have observed that cheeses manufactured with pasteurised goat milk and clotthing with animal and microbial rennet were significantly less hard and chewable compared with other coagulated with vegetable rennet and without differences in springiness and cohesiveness (García et al., 2012). The effect on hardness and chewiness has been especially relevant when the cheese has been coagulated with animal rennet, since the cheeses coagulated without TG showed the highest hardness and chewiness compared with the other coagulants. However, when transglutaminase was added (A + TG), cheeses were least hard and chewable of all. Thus, the samples less hard, less chewable and less spring have been those obtained with animal rennet and TG, vegetal rennet and vegetal rennet with TG. However, they have been the most adhesive, suggesting a strong and negative correlation of adhesiveness with hardness, chewiness and springiness. The adhesiveness has been increased by adding TG in all the samples except in the sample coagulated with microbial rennet. The highest water content of the cheese obtained with animal rennet with TG compared without TG could be responsible for its softer texture, although this was not confirmed by other researchers (Aaltonen, Huumonen, & Myllärinen, 2014). Higher values in the hardness were observed in cheeses with TG compared with control ones using animal rennet as coagulant using an operating temperature of 30 and 34 °C (Özer et al., 2013). The differences with our study can be probably due to the differences in the renneting temperature that was higher in our trial. A greater hardness was observed in soft cheese coagulated with animal rennet and treated with TG, higher cheese yield was also observed (Mahmood & Sebo, 2009). The increase in hardness observed by other authors when adding TG can be probably caused by the dose of TG. It was greater and it was added at the same time as the coagulant or after coagulation. Probably these facts made that the yields obtained by other researchers were different. Literature shows that differences in texture parameters were observed in cheeses made with TG added prior to the rennet compared with others in which the TG was added after the coagulant or at the same time. It was concluded
Fig. 6. Mean values and standard deviation for the TPA parameters (hardness, adhesiveness, springiness, chewiness, springiness and cohesiveness) of cheese coagulated with vegetal rennet without TG (blue bars) and adding TG (orange bars). Different letters show significantly statistical differences between the sample treated with transglutaminase (TG) and the untreated one for the sample coagulated with vegetal rennet.
Fig. 7. Mean values and standard deviation for the TPA parameters (hardness, adhesiveness, springiness, chewiness, springiness and cohesiveness) of cheese coagulated with microbial rennet without TG (blue bars) and adding TG (orange bars). Different letters show significantly statistical differences between the sample treated with transglutaminase (TG) and the untreated one for the sample coagulated with microbial rennet.
showed significantly higher values of Aw. When the effect of the TG treatment has been analyzed the greatest difference in the Aw was observed in the sample coagulated with microbial rennet, with a decrease when the TG was added. Those samples with a greater cheese yield have been those that have shown the highest water content and cohesiveness (Fig. 8), although the Kruskal Wallis test did not find statistically significant differences between samples neither for water content nor for cheese yield. Oppositely to the findings of the current study, an improved yield and a greater water content have been observed in cheese samples coagulated with chymosin and treated with TG compared with a control sample being those differences statistically significant (Pierro et al., 2010). In other studies, no significant differences about water content have been observed when goat's cheeses coagulated with animal rennet were compared with cheeses coagulated using powdered plant coagulant (Pino, Prados, Galán, McSweeney, & Fernández-Salguero, 2009). The slightly lower yield observed for the samples C + TG and V + TG, compared with the respective control samples (C and V) could 6
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Fig. 8. Principal Component Analysis. Cheeses coagulated with animal rennet (A), recombinant chymosin (C), rennet (V) or microbial rennet (M). Cheese treated with microbial transglutaminase (+TG). Water content (W), water activity (Aw).
an effective tool to obtain cheese products with innovative textural properties and slightly improved yields.
that best results in terms of gel formation were obtained when TG was added before the rennet (De sá & Bordignon-Luiz, 2010).
Acknowledgements
4. Conclusions
The authors are grateful to Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (Project # ED431B 2016/ 009). The financial support of Ministerio de Ciencia e Innovación (Spain) for this work (project RTC2014-1835-2) is also acknowledged.
When the rennet source has been studied, the sample coagulated with recombinant chymosin has been the one that has shown a higher yield, while the lowest yield has been obtained using the microbial rennet. Cheeses treated with animal rennet have been the hardest, chewiest and springiest, but they have also been the least springiness and one of the least cohesive. Texture parameters and cheese yield have been affected by the TG treatment. Cheeses coagulated with animal rennet have been the most affected by the TG treatment since all textural parameters have been affected in a significant way. The TG addition with animal rennet caused a significant decrease in hardness, chewiness and springiness. The adhesiveness and cohesiveness values have increased significantly. Incubating with TG before the coagulant addition can prevent great increases in hardness frequently associated with the TG incorporation in other steps of the cheese manufacture, achieving a slight increase in cheese yield in cheeses coagulated with animal and microbial rennet. A previous incubation with TG before the animal rennet addition could be
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Interaction between rennet source and transglutaminase in white fresh cheese production: Effect on physicochemical and textural properties
T
Belén García-Gómeza, Mª Lourdes Vázquez-Odériza, Nieves Muñoz-Ferreirob, Mª Ángeles Romero-Rodrígueza, Manuel Vázqueza,∗ a b
Department of Analytical Chemistry, Faculty of Science, University of Santiago de Compostela, 27002, Lugo, Spain Modestya Research Group, Department of Statistics, Mathematical Analysis and Optimization, University of Santiago de Compostela, 27002, Lugo, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Texture TPA Yield Cheese Rennet Transglutaminase
The aim of this study was to determine the effect of the rennet source and the interaction of rennet with TG on physicochemical and texture properties of fresh cheese made with pasteurized cow milk. The rennets used were: animal, recombinant chymosin, vegetal or microbial rennet. Cheeses with and without addition of TG was obtained. The rennet source did not affect statistically the activity water and water content of the cheese. The highest yield was obtained using recombinant chymosin. The rennet source showed important effects on texture parameters. Microbial and vegetal rennets increased the adhesiveness of cheeses. Cheeses coagulated with animal rennet were the most affected by the TG treatment, observing a significant decrease in hardness (62.19%), chewiness (75.09%) and springiness (43.75%). The adhesiveness and cohesiveness values also increased significantly. An increase of 0.69% in the yield was achieved when the TG was combined with animal rennet. The TG addition combined with animal rennet can be an effective way to obtain cheese products with innovative textural properties and slightly improved yield.
1. Introduction The manufacture of cheese has been a common practice of mankind for thousands of years. It basically involves the conversion of liquid milk into solid curd, followed or not by the biochemical evolution of the curd into ripened cheese (Reis & Malcata, 2011). The traditional manufacture of a cheese could not satisfy the demand for cheese, but the development of refrigeration systems, the commercial availability of the starters and the use of pasteurized milk allowed a qualitative and quantitative leap in cheese production. The implementation of new technologies is necessary to optimize the production process and satisfy the consumer demand (Ghanimah, Hanafy, Hassanein, & Hashim, 2018; Johnson, 2017). In addition to be a widely consumed product due to its sensory properties, it is necessary to highlight the important role in human nutrition throughout the history of humankind. However, dairy products in general and cheese in particular have been associated recently with negative effects on health. The nutritional importance of the cheese was confirmed in a recent study where it was observed that during milk fermentation in cheese manufacture the phospholipid concentration suffer a great increasing enhancing the cheese nutritional
∗
value (Ferreiro & Rodríguez-Otero, 2018; Zhi et al., 2017, 2018). In addition, recent studies suggest that bioactive compounds such as functional lipids (CLA), vitamins, antimicrobial peptides, γ-aminobutyric acid and exopolysaccharides with a potential effect on disease prevention could be released by some lactic acid bacteria (SantiagoLópez et al., 2018). Many researches are being carried out to improve the cheeses from the nutritional point of view without altering the sensorial properties or the shelf life, such as the reduction of the sodium content more than 30% using salt replacers (Carmi & Benjamin, 2017). Most varieties of cheese are produced by enzymatic coagulation with rennet. Cheese has become a very important product in the dairy industry (Fox, Guinee, Cogan, & McSweeney, 2016; Pinheiro et al., 2012). Nowadays functional dairy products present in market are mainly made from cow milk although cheese manufactured with milk of small ruminants as sheep and goats are also frequent (Ardelean, Otto, Jaros, & Rohm, 2012; Llamas-arriba et al., 2019; Pinheiro et al., 2012). 1.0) Enzymatic coagulation of milk is a crucial step in cheesemaking process. Rennet is a mixture of proteolytic and lipolytic enzymes such as chymosin, pepsin and lipase. Its activity is required for enzymatic milk coagulation. Rennet is the responsible of the degradation of casein proteins through disruption of covalent peptide bonds of
Corresponding author. E-mail address: [email protected] (M. Vázquez).
https://doi.org/10.1016/j.lwt.2019.108279 Received 14 May 2019; Received in revised form 7 June 2019; Accepted 17 June 2019 Available online 21 June 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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κ-casein formed between phenylalanine and methionine (Selin et al., 2018). The enzymatic cleavage causes the destabilization of the casein micelles that are aggregated and a three-dimensional protein matrix is obtained forming the curd (Amira, Besbes, Attia, & Blecker, 2017; Fox et al., 2016). The main role of proteolysis is to be responsible of the curd formation. Moreover, it is related with the release of amino acids as precursors of catabolic reactions in which flavor compounds are released (Fox et al., 2016; Hayaloglu & Karabulut, 2013; Katsiari, Alichanidis, Voutsinas, & Roussis, 2000). 2.0) In cheese manufacturing, the coagulation of milk is traditionally made by calf rennet chymosin obtained from the abomasum of nursing animals. Rennet extracted from calf, kid or lamb abomasus were the primary commercial animal source. Other enzymes are capable of initiating the proteolysis like aspartic proteinases. They can be extracted from some plants, microorganism and different ruminants such as pig, lamb, goat and rabbit (Balabanova, Ivanova, & Vlaseva, 2017; Selin et al., 2018). Nowadays, more than 90% of the rennet used in the cheese is fermentation-produced chymosin. The use of substitutional enzymes of calf rennet can be an interesting alternative to meet the demand of lacto-vegetarians people and it has the advantage of being both kosher and halal approved (Jacob, Jaros, & Rohm, 2011; Johnson, 2017). 3.0) Rheological and sensory properties of the cheese are related to the enzymatic activity (Amira et al., 2017). The rennet substitutes must have a relatively weak proteolytic activity in order to maintain the quality of the product since an excessive proteolysis may cause yield losses during manufacture due to denaturing of к-casein in the whey and the development of bitter taste in the final product (Fox et al., 2016; Mazorra-Manzano et al., 2013). For this reason, chymosin is particularly suitable for the cheesemaking due to its low proteolytic activity and its high milk clotting activity. The most important substitutes that meet the requirements of cheese making include microbial, recombinant and plant-based enzymes (Shah & Mir, 2014). Plant proteases are classified into several groups according to the catalytic mechanism used during the hydrolytic process. Many enzymes used as milk coagulants contain aspartic proteases. Enzymes with proteolytic action such as cysteine and serine are also used (Shah & Mir, 2014). Aspartic proteases are extracted mainly from the flowers of many varieties of plant species (Amira et al., 2017). Rennet extracted from dried flowers of the wild thistle Cynara cardunculus has been employed on manufacture of traditional semi-hard cheeses from raw ovine milk at the farm level since ancient times in different Spanish regions (Almeida & Simões, 2018; Sousa & Malcata, 1997). Its application was also studied in cow's milk (Sousa & Malcata, 2002). The best option recommended is the use of cardoon extract formulations (Almeida & Simões, 2018). Many extracellular microbial proteases act similar like chymosin. The advantage of microbial rennet is their availability since it can be easily obtained by fermentation. However, the increased activity of those proteins during cheese making leads to an excessive degradation of proteins. These are lost in the serum and have a negative effect on the cheese yield (Jacob, Jaros, et al., 2011). The excessive proteolytic activity was controlled by chemical modifications and using genetic engineering tools on the microbial organism (Yegin & Dekker, 2013). Nowadays, microbial proteases are obtained from Rhizomucor miehei, Rhizomucor pusillus, and Cryphonectria parasitica (Balabanova et al., 2017; Sumantha, Larroche, & Pandey, 2006; Yegin & Dekker, 2013). They have been established for large-scale production being commercialized and applied in cheese manufacturing (Jacob, Nöbel, Jaros, & Rohm, 2011). The aspartic protease produced by Rhizomucor miehei is the most used in cheese manufacture and is commercially available at different levels of thermostability and purity (Jacob, Jaros, et al., 2011). Therefore, nowadays four sources of rennet are available in the market: calf rennet, recombinant chymosin, vegetal rennet or microbial rennet. To the best of our knowledge, there are not studies comparing
the effect of the four rennets on the same type of cheese. On the other hand, food industry is looking to develop new dairy products with novel physical and functional characteristics (Sharma, Lorenzen, & Qvist, 2001). In the cheese industry, it is also important to improve the yield. Enzymatic crosslinking with transglutaminase (TG) have a great potential to achieve both aims (Cozzolino et al., 2003; Gaspar & De Góes-Favoni, 2015; Kuraishi, Yamazaki, & Susa, 2001; Mahmood & Sebo, 2009; Martins et al., 2014; Taghi Gharibzahedi et al., 2018). TG can change the composition of proteins by crosslinking, amine incorporation and deamidation (García-Gómez, RomeroRodríguez, Vázquez-Odériz, Muñoz-Ferreiro, & Vázquez, 2018, 2019; Motoki & Seguro, 1998). Those reactions modify the functional properties of vegetable and animal proteins allowing to obtain products with improved rheological and sensory properties (Gauche, Vieira, Ogliari, & Bordignon-Luiz, 2008; Yokoyama, Nio, & Kikuchi, 2004). A microbial transglutaminase was isolated from Streptomyces mobaraensis at the late 1980s. It was Ca2+ independent and stable over a wide range of temperatures and pH (Ando et al., 1989). Furthermore, it was particularly useful for food industrial applications because it shows an improved reaction rate, broad substrate specificity for the acyl donor and low-cost mass production (Guerra-Rodríguez & Vázquez, 2014; Jaros, Partschefeld, Henle, & Rohm, 2006). The aim of this study was to determine the effect of the rennet source and the interaction of rennet with microbial transglutaminase on physicochemical and texture properties of fresh cheese made with pasteurized cow milk. 2. Materials and methods 2.1. Raw materials Commercially available pasteurized full fat cow's milk (protein, 3.1 g/100 g; fat, 3.6 g/100 g; ESM, 3.6 g/100 g. Four types of coagulants were used: animal rennet from calf, vegetal rennet from Cynara cardunculus, microbial rennet from Rhizomucor miehei and recombinant chymosin from Kluyveromyces lactis. All coagulants were supplied by Abiasa (Tui, Pontevedra, Spain). TG was produced in our laboratory following the manufacture process described in our Spanish patent ES-2376439 (Vázquez & Guerra-Rodriguez, 2012). A spectrophotometry procedure was used to measure the TG activity before. A calibration curve was made using Lglutamic acid γ-monohydroxamate (Sigma-Aldrich Corp, St. Louis, MO, USA). N-α-CBZ-gln-gly (Sigma-Aldrich Corp, St. Louis, MO, USA) was used as substrate. One unit of TG is defined as the formation of 1 micromol L-glutamic acid γ-monohydroxamate in 1 min at 37 °C (Grossowicz, Wainfan, Borek, & Waelsch, 1950). The measured activity previously to use were 470 U/g. 2.2. Cheese manufacturing The cheeses have been manufactured as described below. The first batch was coagulated with animal rennet (A); the second batch was coagulated with animal rennet and microbial transglutaminase (A + TG); the third batch was coagulated with recombinant chymosin (C); the fourth batch was coagulated with recombinant chymosin and microbial transglutaminase (C + TG), fifth batch was coagulated with vegetal rennet (V); the sixth batch was coagulated with vegetal rennet and microbial transglutaminase (V + TG); the seventh batch was coagulated with microbial rennet (M); and the eight batch was coagulated with microbial rennet and microbial transglutaminase (M + TG). The milk used to make the samples treated with TG (batch 2, 4, 6 and 8) was heated up to 40 °C, then, TG was added (0.8 U/g protein) and the mixture was incubated during 20 min at 38 °C before curd production. The milk used to make the batches without microbial transglutaminase (batch 1, 3, 5 and 7) was heated up to 38 °C. Then with the milk at 38 °C in all the batches, 0.25 mL/kg of coagulant, 2
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Fig. 1. Flow diagram for cheese production treated with transglutaminase (A) and without transglutaminase (B).
maximum height of the first compression. Adhesiveness was measured as the area under x-axis on compression graph curve. Springiness was calculated as the height of the product on the second compression divided by the height of the first peak. Cohesiveness was calculated as the ratio of the areas of the second and the first peak. Chewiness was calculated as hardness*cohesiveness*springiness (Castro-Briones et al., 2009). Eight samples were analyzed for each cheese trial.
0.3 mL/kg of CaCl2 (43.5%) and 6 g/kg of NaCl were added to obtain the curd. The curd obtained during the enzymatic coagulation was cut into cubes having size of 2 cm. The curd was separated from the whey and was placed in perforated cylindrical molds with 12 cm of diameter and 7 cm of height, without putting pressure. The curd was drained during 12 h. Then, cheeses were demolded and stored at 4 °C for at least 24 h before analyze them. Cheeses were sampled for instrumental texture analysis, physico-chemical analysis and yield measurement. The flow diagram of cheese manufacture and the analyses performed is shown in Fig. 1. Duplicate batches were performed.
2.4. Statistical analysis Two-way analysis of variance (ANOVA) was accomplished on the texture parameters considering the source of rennet (A, C, V and M), TG addition (yes/not) and their interaction as fixed sources of variation. It was considering the type of coagulant as treatment 1 with levels A, C, V and M and the TG addition as treatment 2 (yes/not). For the parameters Aw, W and Y, the Kruskal-Wallis test was applied when TG addition effect was analysed and Mann-Whitney U test was applied when the effect of the type of coagulant was studied (Granato, de Araújo Calado, & Jarvis, 2014). Principal Component Analysis (PCA) was applied in order to identify underlying factors (texture parameters, Aw, W and Y) that explain the pattern of correlations within the set of observed variables. The average for each sample for each variable were used to perform the data matrix for PCA. The variables have not the same units. Therefore they were scaled in order to give the same influence to each one (Granato et al., 2018; Le, Josse, & Husson, 2008a). Statistical calculations were performed using IBM SPSS Statistics 20 software for Windows (IBM, Armonk, NY, USA) for ANOVA and R software (R Core
2.3. Analysis Water content (W) was determined gravimetrically drying at 102 °C for 48 h by triplicate. Water activity (Aw) was measured using AquaLab meter (Pullman, WA, USA). W and Aw of fresh cheese were determined in the final product on triplicate samples. Yield (Y) of each batch was expressed as weight of cheese obtained from 100 g of milk employed for the production of cheese. Fresh cheese was cut into small cubes (2 × 2 × 1.5 cm). Texture profile was determined at room temperature using a TA-XTplus texturometer (Stable Micro System, Viena Court, UK). It was used a cylindrical aluminum probe (P/50) with 50-mm diameter. The compression speed was 60 mm/min. Samples were compressed to 60% of the original height. It was measured hardness, adhesiveness, springiness, cohesiveness and chewiness. Hardness was measured as the force at the 3
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Table 1 Results of water activity (Aw), water content and production yield for the cheeses with the rennet studied (no TG added). Aw
Water content (% w/w)
Yield (%)
63.79 63.60 68.02 68.91
17.78 20.19 19.83 16.57
Rennet source Animal Chymosin Vegetal Microbial
0.990 0.990 0.990 0.990
± ± ± ±
0.000 0.003 0.004 0.001
± ± ± ±
0.00 0.03 0.01 0.01
Team, 2018) using the FactoMineR package for the rest of statistical analysis (Le, Josse, & Husson, 2008b). Differences at a level of pvalue < 0.05 were considered significant. 3. Results 3.1. Effect of the rennet source Table 1 shows the results of Aw, water content and production yield. The Aw values did not change with the rennet source, showing values around 0.99. The samples showed similar water contents in cheeses with animal rennet and recombinant chymosin, but lower than those obtained with vegetal and microbial rennets. However, no significant differences were found (p-value > 0.05). Regarding the yield, the lowest yield was obtained using microbial rennet (16.6%) and the highest using recombinant chymosin (20.19%). The effect of the rennet source on cheese texture was determined by the TPA analysis. The mean values and standard deviations of the TPA parameters are given in Figs. 2 and 3. The cheeses obtained with animal rennet were significant harder than the others (p-value ≤0.05) (Fig. 2a). Chymosin and microbial rennet showed intermediate hardness (2672l.46 and 2594.43 g, respectively). The vegetal rennet produced cheeses that are significantly softer than the obtained with the other rennets (p-value ≤ 0.05). The cheeses obtained with animal rennet is a 40.17% harder than that obtained with vegetal rennet (2101.74 g). Cheeses coagulated with animal rennet showed the smallest adhesiveness (−49.96 g s). The highest adhesiveness was measured in the cheeses obtained with microbial rennet (−255.33 g s) (Fig. 2b). Microbial rennet gave cheeses 80.43% more adhesive than those obtained with animal rennet. Significant differences (p-value≤0.05) were found for all the rennets used, except for vegetal and microbial rennets. Microbial and vegetal rennets were similar between them and significantly different compared with chymosin and animal rennets (pvalue ≤ 0.05). Chewiness showed the same trend that hardness (Fig. 2c). The lowest values were observed in cheese coagulated with vegetal rennet and the highest using animal rennet. Cheese obtained with animal rennet showed the highest values for springiness, following by cheeses coagulated with chymosin and microbial rennet (Fig. 3a). Again, the vegetal rennet gave the lowest values. The differences observed in cohesiveness was smaller compared with the other texture parameters (Fig. 3b). The highest values were obtained using vegetal and microbial rennets. The lowest values were measured in samples coagulated with animal rennet and chymosin. Chymosin and animal rennet gave cheese with similar cohesiveness but significantly smaller (p-value≤0.05) than the obtained using vegetal and microbial rennet. The difference between microbial rennet and chymosin was 21.43%.
Fig. 2. Mean values and standard deviation for hardness, adhesiveness and chewiness. Different letters show significative statistical differences between the rennet source (A was coagulated with calf rennet; C was coagulated with recombinant chymosin; V was coagulated with vegetal rennet and M was coagulated with microbial rennet).
cheeses without TG, except in those coagulated with chymosin. The differences in Aw values were statistically significant for samples coagulated with chymosin and vegetable rennet. The addition of TG increased the water content of the samples coagulated with animal rennet and chymosin, 3.8 and 2.7% respectively. No statistically significant differences were found between the sample treated with TG and the untreated sample for any of the coagulants used. When TG was added, the differences observed in the yield were scarce, barely of the 0.5 and 0.7% for the animal and vegetable rennet, respectively. I was observed even a slight descent in the cheeses coagulated with chymosin and vegetable rennet. Fig. 4 show the TPA parameters obtained for the cheese coagulated with animal rennet. The cheeses coagulated with animal rennet with TG showed a decrease in hardness of 62.19%. Regards adhesiveness, the sample coagulated with animal rennet has also been affected by the TG addition (p-value≤0.05). Adhesiveness values decreased from −49.96 to −167.93 g s when TG was added. The chewiness value decreased by
3.2. Effect of the TG addition The four rennets have been studied separately to compare the effect of the transglutaminase addition in each of them (Table 2). The values of Aw were lower in the cheeses treated with TG compared with the 4
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Fig. 4. Mean values and standard deviation for the TPA parameters (hardness, adhesiveness, springiness, chewiness, springiness and cohesiveness) of cheese coagulated with animal rennet without TG (blue bars) and adding TG (orange bars). Different letters show significantly statistical differences between the sample treated with transglutaminase (TG) and the untreated one for the sample coagulated with calf rennet.
Fig. 3. Mean values and standard deviation for springiness and cohesiveness. Different letters show significative statistical differences between the rennet source (A was coagulated with calf rennet; C was coagulated with recombinant chymosin; V was coagulated with vegetal rennet and M was coagulated with microbial rennet). Table 2 Results of water activity (Aw), water content and yield for the samples with TG treatment. The asterisk shows significant statistical differences comparing rennet source when the transglutaminase was used. Rennet source
Aw
Animal Chymosin Vegetal Microbial
0.987 0.991 0.989 0.986
± 0.001 ± 0.002* ± 0.002* ± 0.002
Water content (% w/w)
Yield (%)
67.59 66.31 67.54 68.81
18.47 19.61 17.84 17.08
± ± ± ±
0.04 0.13 0.03 0.01
Fig. 5. Mean values and standard deviation for the TPA parameters (hardness, adhesiveness, springiness, chewiness, springiness and cohesiveness) of cheese coagulated with chymosin without TG (blue bars) and adding TG (orange bars). Different letters show significantly statistical differences between the sample treated with transglutaminase (TG) and the untreated one for the sample coagulated with recombinant chymosin.
75.09% when TG was added. The highest value for the springiness was that observed for the sample coagulated with animal rennet without TG. When TG has been added, springiness decreased by 43.75%. The TG addition were responsible of an increase in cohesiveness around 11.11% in the samples treated with animal rennet (A + TG) compared with the untreated sample (A). Fig. 5 show the TPA parameters obtained for the cheese coagulated with recombinant chymosin. Neither the hardness nor the adhesiveness of cheeses coagulated with recombinant chymosin have been affected by the addition of TG, obtained values around 2670 g and −120 g s, respectively. Opposing to what happened with animal rennet, the incorporation of TG in the manufacture of cheeses coagulated with recombinant chymosin significantly increases chewiness and springiness (Fig. 5). The observed increase was 19.19% for chewiness, 9.09% for springiness. The cohesiveness was also increased using TG like in the case of using animal rennet. Fig. 6 show the TPA parameters obtained for the cheese coagulated with vegetal rennet. Treatment with TG slightly increased all the texture parameters. The increase was lower than 10%, except for the cohesiveness that decreased by 16%. However, the differences were not
significantly. Fig. 7 show the TPA parameters obtained for the cheese coagulated with microbial rennet. The treatment with TG has been responsible for a decrease in all the texture parameters. The decrease in texture values were statistically significant for chewiness, springiness and cohesiveness. Chewiness was the parameter more affected by the addition of TG with a decrease of 21.18%. Finally, Principal Component Analysis was performed in order to facilitate the joint vision of the physicochemical variables, TPA parameters and the yield obtained for the different samples. The explained variance of the first and second factor accounts for 80.16%. Fig. 8 shows that the sample with a higher yield and Aw has been coagulated with recombinant chymosin without transglutaminase (C). The lowest yields and Aw have been observed in the samples coagulated with microbial rennet (M and M + TG). When the samples were treated with TG, the differences in yields comparing the different rennets were minimized. Comparing the rennet source, the sample coagulated with chymosin 5
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be due to the fact that the preincubation of the milk with the TG would have inhibited hydrolysis of k-casein during the coagulation, inhibiting the micelles aggregation. Consequently, the milk coagulation was affected (Bönisch, Heidebach, & Kulozik, 2008). Probably due to this fact, some researchers using chymosin as coagulant did not get the coagulation of the milk when incubated the milk with TG for 20 min at 20 °C before adding the coagulant. However, an increasing in cheese yield was observed when the transglutaminase was added after the clotting enzyme inducing the formation of the coagulum (Cozzolino et al., 2003). Similar results to those obtained by the latter researchers were observed in other study (Yüksel, Avci, & Erdem, 2011). However, milk coagulation was achieved by incubating with TG prior to adding the coagulant although an increase in renneting time was observed (Domagała et al., 2016). In other researches, the increase in yield achieved in TG treated white-brined cheese coagulated with animal rennet was attributed not only to a higher serum retention related with the alteration of the physical properties of the casein gels with a lower pore size but also a slight increase of protein fraction observed (Özer, Hayaloglu, Yaman, Gürsoy, & Şener, 2013). Our results show a strong correlation between water content and cohesiveness (Fig. 8). The sample coagulated with chymosin (C), it has been the least cohesive and with a lower water content. The cohesiveness values observed in the samples coagulated with animal rennet have been greater when transglutaminase has been added (Fig. 5). Those results were according with the findings of other researchers (Özer et al., 2013). The highest values of hardness, springiness and chewiness were observed in the sample coagulated with animal rennet. When this coagulant is combined with TG, a decrease in the values of those parameters was observed. Opposite our findings, other researchers have observed that cheeses manufactured with pasteurised goat milk and clotthing with animal and microbial rennet were significantly less hard and chewable compared with other coagulated with vegetable rennet and without differences in springiness and cohesiveness (García et al., 2012). The effect on hardness and chewiness has been especially relevant when the cheese has been coagulated with animal rennet, since the cheeses coagulated without TG showed the highest hardness and chewiness compared with the other coagulants. However, when transglutaminase was added (A + TG), cheeses were least hard and chewable of all. Thus, the samples less hard, less chewable and less spring have been those obtained with animal rennet and TG, vegetal rennet and vegetal rennet with TG. However, they have been the most adhesive, suggesting a strong and negative correlation of adhesiveness with hardness, chewiness and springiness. The adhesiveness has been increased by adding TG in all the samples except in the sample coagulated with microbial rennet. The highest water content of the cheese obtained with animal rennet with TG compared without TG could be responsible for its softer texture, although this was not confirmed by other researchers (Aaltonen, Huumonen, & Myllärinen, 2014). Higher values in the hardness were observed in cheeses with TG compared with control ones using animal rennet as coagulant using an operating temperature of 30 and 34 °C (Özer et al., 2013). The differences with our study can be probably due to the differences in the renneting temperature that was higher in our trial. A greater hardness was observed in soft cheese coagulated with animal rennet and treated with TG, higher cheese yield was also observed (Mahmood & Sebo, 2009). The increase in hardness observed by other authors when adding TG can be probably caused by the dose of TG. It was greater and it was added at the same time as the coagulant or after coagulation. Probably these facts made that the yields obtained by other researchers were different. Literature shows that differences in texture parameters were observed in cheeses made with TG added prior to the rennet compared with others in which the TG was added after the coagulant or at the same time. It was concluded
Fig. 6. Mean values and standard deviation for the TPA parameters (hardness, adhesiveness, springiness, chewiness, springiness and cohesiveness) of cheese coagulated with vegetal rennet without TG (blue bars) and adding TG (orange bars). Different letters show significantly statistical differences between the sample treated with transglutaminase (TG) and the untreated one for the sample coagulated with vegetal rennet.
Fig. 7. Mean values and standard deviation for the TPA parameters (hardness, adhesiveness, springiness, chewiness, springiness and cohesiveness) of cheese coagulated with microbial rennet without TG (blue bars) and adding TG (orange bars). Different letters show significantly statistical differences between the sample treated with transglutaminase (TG) and the untreated one for the sample coagulated with microbial rennet.
showed significantly higher values of Aw. When the effect of the TG treatment has been analyzed the greatest difference in the Aw was observed in the sample coagulated with microbial rennet, with a decrease when the TG was added. Those samples with a greater cheese yield have been those that have shown the highest water content and cohesiveness (Fig. 8), although the Kruskal Wallis test did not find statistically significant differences between samples neither for water content nor for cheese yield. Oppositely to the findings of the current study, an improved yield and a greater water content have been observed in cheese samples coagulated with chymosin and treated with TG compared with a control sample being those differences statistically significant (Pierro et al., 2010). In other studies, no significant differences about water content have been observed when goat's cheeses coagulated with animal rennet were compared with cheeses coagulated using powdered plant coagulant (Pino, Prados, Galán, McSweeney, & Fernández-Salguero, 2009). The slightly lower yield observed for the samples C + TG and V + TG, compared with the respective control samples (C and V) could 6
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Fig. 8. Principal Component Analysis. Cheeses coagulated with animal rennet (A), recombinant chymosin (C), rennet (V) or microbial rennet (M). Cheese treated with microbial transglutaminase (+TG). Water content (W), water activity (Aw).
an effective tool to obtain cheese products with innovative textural properties and slightly improved yields.
that best results in terms of gel formation were obtained when TG was added before the rennet (De sá & Bordignon-Luiz, 2010).
Acknowledgements
4. Conclusions
The authors are grateful to Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (Project # ED431B 2016/ 009). The financial support of Ministerio de Ciencia e Innovación (Spain) for this work (project RTC2014-1835-2) is also acknowledged.
When the rennet source has been studied, the sample coagulated with recombinant chymosin has been the one that has shown a higher yield, while the lowest yield has been obtained using the microbial rennet. Cheeses treated with animal rennet have been the hardest, chewiest and springiest, but they have also been the least springiness and one of the least cohesive. Texture parameters and cheese yield have been affected by the TG treatment. Cheeses coagulated with animal rennet have been the most affected by the TG treatment since all textural parameters have been affected in a significant way. The TG addition with animal rennet caused a significant decrease in hardness, chewiness and springiness. The adhesiveness and cohesiveness values have increased significantly. Incubating with TG before the coagulant addition can prevent great increases in hardness frequently associated with the TG incorporation in other steps of the cheese manufacture, achieving a slight increase in cheese yield in cheeses coagulated with animal and microbial rennet. A previous incubation with TG before the animal rennet addition could be
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