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Exopolysaccharide-producing culture in the manufacture of Prato cheese
LWT - Food Science and Technology 72 (2016) 383e389
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Exopolysaccharide-producing culture in the manufacture of Prato cheese Raquel Sant’Ana Coelho Nepomuceno a, Luiz Carlos Gonçalves Costa Junior b, Renata Golin Bueno Costa b, * a
Barbosa & Marques S/A, R. Aluísio Pereira Esteves, 250, 35032-010, Governador Valadares, Minas Gerais, Brazil ria de Minas Gerais, Instituto de Laticínios Ca ^ndido Tostes, R. Tenente Luiz de Freitas, 116, 36045-560, Juiz de Fora, Minas Empresa de Pesquisa Agropecua Gerais, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2015 Received in revised form 25 April 2016 Accepted 26 April 2016 Available online 3 May 2016
Exopolysaccharide (EPS)-producing culture in capsular form has been used in the manufacture of Prato cheese to evaluate the effect on the physicochemical and sensory characteristics, texture properties, melting capacity, and yield of Prato cheese. Prato cheeses were manufactured with and without the addition of EPS-producing culture, and assessed within 120 days of refrigerated storage. The addition of EPS-producing culture increased yield in 0.25 kg/100 kg of milk when compared to the cheese made with EPS non-producing culture. Cheeses manufactured with EPS-producing culture had higher moisture content than the control; however, this did not affect proteolysis, pH, and the melting capacity of cheeses. In addition, cheeses containing EPS had lower hardness and chewiness than the control cheese. Although EPS has changed the texture characteristics, it did not affect the sensory acceptance, which was similar for both treatments. These results indicate that Prato cheeses made with EPS-producing culture have better performance without changing their physicochemical and sensory characteristics. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Prato cheese Brazilian cheese Capsular exopolysaccharide Cheese yield
1. Introduction Exopolysaccharide (EPS)-producing cultures (EPSþ) have been used in cheese making due to the excellent property of EPS to bind water and retain moisture. In low fat cheeses, EPS improves texture and quality, and it becomes similar to its full-fat cheese (Abou Ayana & Ibrahim, 2015; Lynch, McSweeney, Arendt, UniackeLowe, & Coffey, 2014). Encapsulated EPS-producing strains can be used to increase moisture in cheese and improve the melting property of low fat mozzarella, without adversely affecting the whey viscosity, with no changes in cheese properties and processing (De Vuyst et al., 2003; Zisu & Shah, 2007). In reduced fat Cheddar cheese, the use of Lactococcus lactis ssp. cremoris producing EPS in capsular form led to an increase in moisture content and cheese yield, and improved cheese firmness and elasticity after six months of ripening (Dabour, Kheadr, Benhamou, Fliss, & Lapointe, 2006). Previous studies using EPS-producing culture in low fat cheese found an increase in cheese yield due to the higher EPS
* Corresponding author. E-mail address: [email protected] (R.G.B. Costa). http://dx.doi.org/10.1016/j.lwt.2016.04.053 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
water retention capacity (Costa et al., 2010; Di Cagno, De Pasquale, De Angelis, Buchin, Rizzello, & Gobbetti, 2014; Oluk, Güven, & Hayaloglu, 2014; S¸anli, Gursel, S¸anli, Acar, & Benli, 2013). Although several authors have studied low fat cheeses, there are few studies on the effect of EPS-producing cultures in cheese jera, Guerrer and Garibay (2009) without fat reduction. Guzman, Na evaluated the use of EPS-producing Streptococcus thermophilus in the manufacture of Panela Mexican cheese, a soft and moist cheese. These authors have reported higher water retention in cheese, thus rrez-Me ndez, Sepulveda, increasing yield. Trancoso-Reyes, Gutie ndez-Ochoa (2014) also found higher yield and moisand Herna ture and fat retention in Chihuahua cheese made with EPSproducing bacteria. In Brazil, Prato cheese and its varieties correspond about 20% of all cheeses produced in the country (Abiq, 2011). Prato cheese is of Danish origin, similar to Gouda and Danbo, despite its different flavor and texture. It is consumed both directly and indirectly (Cichoscki, Valduga, Valduga, Tornadijo, & Fresno, 2002). Although it has a great representativeness in Brazilian market, there are no studies using EPS-producing culture in the manufacture of Prato cheese. Therefore, the aim of this study was to evaluate the effect of capsular EPS-producing culture on the physicochemical and
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culture used in Prato cheese manufacture was used, consisting of Streptococcus thermophilus, Lactococcus lactis ssp. lactis and/or Lactococcus lactis ssp. cremoris (Lyofast MOS 062 E) (Clerici-Sacco, Caglifio Clerici, Cadorago, Italy), at a dose of 50 UC (77.5 g) for 12,500 L of milk. Analyses of proteolysis, pH, melting capacity, and texture profile were performed after 2, 30, 60, 90, and 120 days of refrigerated storage in all 5 replicates. 2.2. Physicochemical characterization The cheeses were characterized after two days of refrigerated storage in all 5 replicates for total solids, fat levels, protein, and salt content according to IDF International Standards 4A (1982), 5B (1986), 20B (1993) and 88A (1988), respectively. The pH was determined according to AOAC (1980) and total nitrogen (NT) by Kjeldahl method (AOAC, 1995). The pH 4.6- soluble nitrogen and 12% TCA-soluble nitrogen were determined according to Barbano, Lynch, and Fleming (1991), and AOAC (1995), respectively. Proteolysis was evaluated by the extent and depth of proteolysis indexes using the following equations:
Extent of proteolysis ¼ ð% pH 4:6 soluble nitrogen=% total nitrogenÞ 100 (1) Depth of Proteolysis ¼ ð% 12% TCA soluble nitrogen=% total nitrogenÞ 100 (2)
Fig. 1. Flowchart of Prato cheese manufacture.
sensory characteristics, texture properties, melting capacity, and yield of Prato cheese. 2. Material and methods 2.1. Manufacture of Prato cheese Prato cheese was manufactured in 5 replicates on different days. The manufacturing process is described in Fig. 1. Each batch of cheese was made with 12,500 L capacity. Calcium chloride (50% w/ v, 0.30 mL/L milk); annatto dye (0.025 mL/100 L milk - Chr. Hansen Brazil, Valinhos, Brazil), starter culture (UC 50, Unita Cento) and liquid microbial coagulant, 100% chymosin (Clerici-Sacco, Caglifio
The pH of the samples and the proteolysis indexes were determined after 2, 30, 60, 90, and 120 days of refrigerated storage in 5 replicates. Milk was analyzed for fat content by the Gerber method (British Standards Institution, 1989) and total nitrogen (AOAC International, 2006). The protein content was determined by multiplying the total nitrogen content by 6.38. The whey was collected 15 min after cutting the curd, and analyzed for fat and total nitrogen contents according to the methods previously mentioned. Actual yield was expressed as kg cheese/100 kg milk. Fat and protein recoveries were calculated according to Alves, Merheb-Dini, Gomes, Silva, and Gigante (2013). Adjusted yield (Yadj) was calculated considering the desired moisture content of 43% (w/w) and the desired NaCl level of 1.6% (w/w) according to equation (3):
Yadj ¼ fYact ½100 ð%realmoisturecontent þ %realsaltcontentÞg=½100 ð%desirablemoisturecontent þ %desirablesaltcontent
Clerici, Cadorago, Italy) were used to manufacture the cheese. The following treatments were carried out: (1) lyophilized capsular exopolysaccharide-producing culture (EPS culture) for direct inoculation and (2) a traditional culture used in Prato cheese manufacture (control). EPS culture was added to the production tank at a dose of 20 Unita Cento (UC) (49.0 g) for Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris (Lyofast MO 242) and 30 UC (28.5 g) for Streptococcus thermophilus (Lyofast ST 4.40) (ClericiSacco, Caglifio Clerici, Cadorago, Italy). As a control, a traditional
(3)
2.3. Melting capacity of Prato cheese The melting capacity was determined by the modified method of Schreiber (Kosikowski, 1982), in quadruplicate. A cheese cylinder of approximately 36 mm in diameter and 7 mm thick was sampled, and discs were cut with the aid of a slicer. Four slices were sampled from the inner region of the cheeses. Each slice was placed in the
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center of a Petri plate, divided into 8 areas of equal diameters. Four diameters were measured for each sample (Di), and then the plates were placed in an oven at 130 C for 10 min (Nonogaki et al., 2007). These authors studied the best time and temperature condition for maximum melting of Prato cheese, without burning the product. After 10 min, the plates were maintained for 30 min at room temperature and the diameters of each melted sample was measured again (Df). The melting capacity was calculated using the equation:
MCð%Þ ¼
. Df 2 Di2 100 Di2
(4)
where: MC: melting capacity; Df: final diameter; Di: initial diameter. 2.4. Texture profile analysis The texture profile analysis (TPA) of the samples was performed in a CT3 Texture Analyzer (Brookfield, Middleboro, USA) using a 4500 g load cell. The cheeses were cut into cylinders of 20 mm diameter and 25 mm in height, placed in sealed plastic bags (to avoid dehydration) and stored at 10 C for 1 h. Samples were taken from the middle of the cheese block to avoid surface effects. The speed of 1 mm/min was used at 40% compression test, using an acrylic cylindrical probe of 25.4 mm diameter and 35 mm height. 2.5. Sensory evaluation Acceptance test was performed after 30 days of refrigerated storage. Two hundred and fifty untrained assessors (fifty consumers on each sampling day) consisting of students, teachers and staff from Instituto de Laticínios Candido Tostes, of varied ages and both sexes, were selected based on regular consumption of cheese, time availability and interest. The acceptance test was evaluated by a 9-point hedonic scale (1 ¼ extremely disliked, 7 ¼ liked moderately, and 9 ¼ liked extremely; Meilgaard, Civille, & Carr, 1999) for the attributes appearance, flavor, texture, and overall acceptance. Cheese samples were cut into 2.5 2.5 2.5 cm pieces and codified with random 3-digits numbers. 2.6. Statistical analysis Statistical analyses were performed using Minitab 12.1 program
Table 1 Physicochemical composition of Prato cheese made with and without addition of EPS-producing culture after two days of refrigerated storage (means ± SD; n ¼ 5). Composition
Moisture (% w/w) Fat (% w/w) Protein (%w/w) NaCl (%w/w) FDMb (%w/w) Salt in moisture (% w/w) MNFSc Total yield (kg cheese/100 kg milk) Adjusted yieldd (kg cheese/100 kg milk) Fat recovery in cheese Protein recovery in cheese
Culturea
P-value
EPS
EPSþ
41.03 ± 0.94 31.74 ± 1.06 23.40 ± 0.44 0.57 ± 0.24 53.82 ± 0.95 1.37 ± 0.55 60.11 ± 0.49 11.15 ± 0.09 10.37 ± 0.06 92.14 ± 0.42 79.39 ± 1.26
43.86 ± 1.44 29.80 ± 0.45 22.25 ± 1.19 0.87 ± 0.18 53.10 ± 0.65 1.94 ± 0.38 62.47 ± 1.68 11.40 ± 0.20 11.07 ± 0.43 91.64 ± 0.70 78.53 ± 0.68
0.01 0.01 0.07 0.13 0.24 0.18 0.03 0.02 0.03 0.25 0.20
a EPS ¼ cheese manufactured with non-EPS-producing starter; EPSþ ¼ cheese manufactured with EPS-producing starter. b FDM ¼ fat in dry matter. c MNFS ¼ moisture in nonfat substance. d Adjusted yield, considering 43% moisture and 1.6% salt.
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for Windows (Minitab Inc., State College). The effect of culture (2 levels), time of refrigerated storage (5 levels), and their interactions were analyzed by ANOVA and Tukey’s test at a significance level of P < 0.05. 3. Results and discussion 3.1. Physicochemical characterization Both Prato cheeses made with the addition of EPS-producing culture and with conventional procedure (Table 1) met the standards established by Brazilian legislation for moisture content (36% and 45.9% w/w) and fat in the dry matter (45% and 59.9% w/w) rio da Agricultura do Brasil, 1997). (Ministe The use of EPS led to an increase in moisture levels (P < 0.05), due to its water retention capacity. Although the water retention is higher for the EPS-culture in ropy form when compared to the capsular form (Dabour, Kheadr, Fliss, & Lapointe, 2005; Petersen, Dave, McMahon, Oberg, & Broadbent, 2000), a greater water retention was observed in the present study using the capsular form, with an increase in moisture content of 6.89% (w/w). Studies on low fat cheese with the addition of exopolysaccharideproducing culture have also found high moisture levels. In Mozzarella cheese, an increase in moisture content was observed for the samples with EPS-producing cultures in ropy form and capsular form, with values of 6.1% and 2.7%, respectively (Petersen et al., 2000), while a higher water retention of 3.6%e4.8% was observed in low fat Cheddar cheese (Dabour et al., 2006). The increase in moisture levels leads to lower dry extract, with significant changes in cheese fat content, which was lower in the cheeses produced with EPSþ (P < 0.05). However, no significant difference was observed in fat retention between treatments (P > 0.05). This difference in fat content was due the dilution effect of the dry extract constituents (Rynne et al., 2007). Half-fat Cheddar cheese made with EPS-producing strain also had higher moisture and less fat levels than the control (Costa et al., 2010). Moisture in nonfat substance (MNFS) made with EPS-producing culture was higher than the control (P < 0.05). The EPS has the ability to bind water through hydrogen bonds (Costa et al., 2010), increasing MNFS without need to change the cheese making process (Hassan, 2008). Cheeses produced with EPS-producing culture have better actual and adjusted yield than the control cheese (P < 0.05). An increase of 0.25 kg cheese/100 kg of milk in yield of Prato cheese produced with EPSþ was observed, which is extremely important, considering that this calculation is performed to estimate the manufacturing costs of dairy products. The experimental results are consistent with those obtained by Dabour et al. (2006) for reducedfat Cheddar cheese, who reported an increase in yield of 0.28e1.19 kg of cheese/100 kg milk for cheeses produced with EPSproducing strains when compared to the control cheese. However, these values are far below the increase of 1.89 kg per 100 kg milk obtained for the Panela Mexican cheese (Guzman et al., 2009), which has high moisture for being a fresh cheese. In addition, Panela cheese was produced with EPS-producing strain in ropy form, which has higher water retention capacity than the capsular form used in Prato cheese of this study (Dabour et al., 2005; Petersen et al., 2000). The use of EPS-producing bacteria in Chihuahua cheese increased moisture and actual yield (TrancosoReyes et al., 2014). Cheeses made with EPS-producing cultures can present distinct characteristics depending on the variety and nature of the culture (Trancoso-Reyes et al., 2014). No significant differences were observed in fat and protein recovery for all treatments (P > 0.05). The difference in yield was due to the higher moisture levels in cheeses made with EPSþ.
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Table 2 pH and proteolysis indexes of Prato cheese with and without addition of EPS-producing culture for 120 days of refrigerated storage. Cheese
Storage time (days)
pH
Extent of proteolysis index (%)
Depth of proteolysis index (%)
EPSþa
2 30 60 90 120
5.32 5.19 5.23 5.28 5.29
± ± ± ± ±
0.09a 0.16b 0.07b 0.08a 0.03a
6.26 ± 1.10c 6.48 ± 2.91c 7.71 ± 3.55ab 14.69 ± 3.24ab 15.58 ± 3.92a
3.27 ± 0.21b 5.26 ± 0.96b 4.80 ± 1.99b 6.08 ± 1.21b 16.57 ± 7.98a
EPS
2 30 60 90 120
5.32 5.18 5.25 5.31 5.37
± ± ± ± ±
0.09a 0.08b 0.06ab 0.03a 0.05a
7.14 ± 1.97b 8.25 ± 4.67b 8.68 ± 5.15b 13.83 ± 2.00ab 19.97 ± 4.05a
2.47 ± 0.61b 6.81 ± 1.08b 3.73 ± 1.33b 5.52 ± 0.65b 17.19 ± 7.71a
a,b a
Means within the same column and the same treatment with different superscript letters differ significantly (P < 0.05); n ¼ 5. EPSþ ¼ cheese manufactured with EPS producing starter; EPS ¼ cheese manufactured with non-EPS-producing starter.
Fig. 2. Melting capacity of Prato cheese manufactured with EPS-producing culture (EPSþ) and without EPS (EPS) subjected to 130 C/10 min during ripening. abMeans between d 2 to 120 within a cheese type with different letters are significantly different (P < 0.05; n ¼ 5).
3.2. Evolution of pH values and proteolysis indexes No significant changes in pH were observed in Prato cheeses manufactured with EPS-producing culture (P > 0.05). The average
pH values of the cheeses with and without EPS over the 120 days of ripening were 5.28 and 5.32, respectively. In preliminary studies (not shown), the EPS-producing culture was added at the beginning of the manufacture, during tank filling, resulting in rapid decrease of pH, altering the final composition of the cheese. Higher moisture levels in cheeses are due to the greater amount of lactose retained in curd, leading to bacteria growth thus lowering the pH (Walstra, Geurts, Noomen, Jellema, & Van Boekel, 1993). The pH interferes with curd syneresis, besides affecting moisture and enzyme activity during ripening. The reduction of pH affects the rheological properties, melting capacity, and elasticity of cheeses (Fox, 2004, Chap. 1). In cheeses manufactured with EPSþ, there was a reduction in time required to reach pH when compared to the control cheese (Dabour et al., 2006; Dabour et al., 2005). In this study, it was necessary to change the moment of addition of the culture to better control the pH drop during the manufacturing process, so that these values were similar between treatments to avoid interference with the cheese characteristics. Therefore, the culture inoculation was carried out when the tank was filled with milk until at least 80% of its capacity. For non-EPS-producing culture, the inoculation occurred when milk has reached 30% of the tank capacity. At the beginning of ripening, a decrease in pH of the cheese was observed (Table 2), which may have been caused by the fermentation of residual lactose and consequent formation of lactic acid (McSweeney & Sousa, 2000). As expected, there was an increase in pH of the cheeses during ripening due to partial degradation of lactic acid and formation of alkali nitrogenous compounds (Salaün,
Fig. 3. Changes in the rheological properties: hardness (A) and chewiness (B) during ripening of Prato cheese manufactured with (-) and without (B) EPS-producing culture (n ¼ 5).
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Mietton, & Gaucheron, 2005). No significant difference was observed for the extent and depth of proteolysis indexes between treatments (P > 0.05). Although there was an increase in moisture content of the cheese manufactured with EPS-producing culture, no changes were observed in proteolysis. Guzel-Seydim, Sezgin, and Seydim (2005) found that the exopolysaccharide binds strongly to water within the cheese matrix, retaining water for a longer time when compared to control. Even the high moisture content (62.2%) in low-fat mozzarella was stabilized within the cheese matrix (Perry, McMahon, & Oberg, 1997). However, a significant effect of time was observed (P < 0.05) for the extent and depth (Table 2) of proteolysis. The increase in proteolysis during ripening is consistent with other studies (Awad, Hassan, & Muthukumarappan, 2005; Dabour et al., 2006; Sallami, Kheadr, Fliss, & Vuillemard, 2004), due casein degradation by the residual coagulant, forming peptides of high molecular weight and the action of microorganisms in cheese, with greater effects over time (McSweeney, 2004). In the manufacture of Prato cheese, similar lactic acid bacteria concentrations and strains were used, as well as the type and amount of coagulant, differing only in the ability of the bacteria to produce EPS. 3.3. Melting capacity Melting is an important parameter in Prato Cheese, due to its ability to change the original shape when subjected to heat. No significant differences were observed in melting properties of the cheeses made with EPSþ (P > 0.05). During ripening, a significant difference was observed in melting capacity (P < 0.05) (Fig. 2), once proteolysis leads to hydration of the para-casein matrix (Lawrence, Creamer, & Gilles, 1987). In addition, the pH strongly affects moisture-protein interactions and hence the melting of the cheese (Kindstedt, Zielinski, & Almena-Aliste, 2001). Considering that no significant difference was observed in pH between treatments, this parameter did not affect melting capacity. Studies have reported better melting properties in low-fat Mozzarella made with EPS-producing culture due to higher moisture content (Merrill, Oberg, & McMahon, 1994; Zisu & Shah, 2007). However, Zisu and Shah (2006) found no significant difference in melting of cheese produced with capsular EPS-producing culture, which is the same type of EPS used in Prato cheese of this study. 3.4. Texture profile analysis The control cheese (EPS) showed higher firmness when compared to the cheese produced with EPS-producing culture (P < 0.05), as reported by other authors (Awad et al., 2005; Dabour et al., 2006). Cheese produced with EPS-producing culture have more open and porous structure due to the EPS water retention capacity (Costa et al., 2010; Hassan & Awad., 2005) as compared to the cheeses made with non-EPS-producing culture, which have a more dense and compact matrix (Costa et al., 2010). The exopolysaccharide interferes with proteineprotein interactions, reducing cheese firmness (Hassan, 2008) and fills empty spaces in protein matrix (Ayala-Hernandez, Goff, & Corredig, 2008). During ripening, a significant reduction in cheese hardness was observed (P < 0.05) (Fig. 3) due to proteolysis of the protein network by chymosin and hydration of proteins (Zisu & Shah, 2007). There is an interdependence between chewiness and hardness (Irudayaraj, Chen, & McMahon, 1999), and in this study chewiness was lower in cheeses made with EPS-producing culture (P < 0.05). Ahmed, El Soda, Hassan, and Frank (2005) also found lower hardness and chewiness values in Karish cheeses made with EPS-
Fig. 4. Cohesiveness, adhesiveness, and springiness of Prato cheese during ripening: presented data show the values for all cheeses manufactured with and without EPSproducing culture (n ¼ 5).
Table 3 Sensory scores for the cheese manufactured without EPS (control) and with EPSproducing culture after 30 days of refrigerated storage (mean ± SD, n ¼ 5). Attributes
EPSþa
Appearance Flavor Texture Overall acceptance
7.58 7.43 7.33 7.42
± ± ± ±
EPS 1.08 1.37 1.31 1.32
7.54 7.28 7.31 7.17
P-value ± ± ± ±
1.03 1.22 1.29 1.12
0.73 0.28 0.91 0.27
a EPSþ ¼ cheese manufactured with EPS producing starter; EPS ¼ cheese manufactured with non-EPS-producing starter.
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producing culture when compared to those without EPS. No significant differences were observed for the parameters cohesiveness, springiness, and adhesiveness of the cheeses between treatments (P > 0.05). During ripening, a significant effect was observed in all texture characteristics (P < 0.05) (Fig. 4). The texture parameters during ripening are influenced by the proteolytic action of the residual coagulant and lactic acid bacteria (Guinee, 2003; Gunasekaran & Mehmet, 2004). The present results are similar to other studies (Awad et al., 2005; Costa et al., 2010; Dabour et al., 2006). 3.5. Sensory evaluation No significant differences were observed in the acceptance test for all treatments (P > 0.05) for the evaluated parameters (Table 3) after 30 days of refrigerated storage. As the EPS-producing culture did not affect pH and proteolysis, which can interfere with the sensory characteristics of Prato cheese, the untrained assessors did not notice the differences between treatments. Although cheeses containing EPS were less hard and chewy in the texture profile analysis, these characteristics did not affect the sensory acceptance, according to the untrained assessors. 4. Conclusions The use of exopolysaccharide-producing culture in capsular form in the manufacture of Prato cheese improved yield and increased the moisture content. The addition of EPS-producing culture did not affect proteolysis, pH, melting capacity and sensory acceptance. However, differences were observed in the rheological parameters hardness, chewiness. The addition of EPS-producing culture in semi-hard cheeses is a promising alternative to increase yield without changing their physicochemical and sensory characteristics. Acknowledgements The authors thank the financial support to Barbosa & Marques S/ A, Sacco-Brasil, and EPAMIG. References Abou Ayana, I. A. A., & Ibrahim, A. E. (2015). Attributes of low-fat yogurt and Kareish cheese made using exopolysaccharides producing lactic acid bacteria. American Journal of Food Technology, 10, 48e57. Ahmed, N. H., El Soda, M., Hassan, A. N., & Frank, J. (2005). Improving the textural properties of an acid-coagulated (Karish) cheese using exopolysaccharide producing cultures. LWT - Food Science and Technology, 38, 843e847. Alves, L. S., Merheb-Dini, C., Gomes, E., Silva, R. da, & Gigante, M. L. (2013). Yield, changes in proteolysis, and sensory quality of Prato cheese produced with different coagulants. Journal of Dairy Science, 96, 7490e7499. AOAC (Association of Analytical Chemists). (1995). Official methods of analysis of AOAC international. Washington: AOAC. AOAC (Association of Official Analytical Chemists). (1980). Hydrogen-ion activity (pH). In W. Horwitz (Ed.), Official methods of analysis (p. 213). Washington: AOAC. AOAC International. (2006). Official methods of analysis (18th ed.). Arlington, VA: AOAC International. ~o brasileira de pro~o Brasileira das Indústrias de Queijo. (2011). Produça Associaça cteos e estabelecimentos sob inspeça ~o federal. Sa ~o Paulo (N~ dutos la ao paginado). Awad, S., Hassan, A. N., & Muthukumarappan, K. (2005). Application of exopolysaccharide-producing cultures in reduced-fat cheddar cheese: texture and melting properties. Journal of Dairy Science, 88, 4204e4213. Ayala-Hernandez, I., Goff, H. D., & Corredig, M. (2008). Interactions between milk proteins and exopolysaccharides produced by Lactococcus lactis observed by scanning electron microscopy. Journal of Dairy Science, 91, 2583e2590. Barbano, D. M., Lynch, J., & Fleming, J. R. (1991). Direct and indirect determination of true protein content of milk by Kjeldahl analysis: collaborative study. Journal of the AOAC, 74, 281e288. British Standards Institution. (1989). Determination of fat content of milk and milk products (Gerber method)dMethods. London, UK: British Standards Institution. Cichoscki, A. J., Valduga, E., Valduga, A. T., Tornadijo, M. E., & Fresno, J. M. (2002).
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Mehmet (Eds.), Cheese rheology and texture (pp. 299e324). Boca Raton: CRC Press. Guzel-Seydim, Z. B., Sezgin, E., & Seydim, A. C. (2005). Influences of exopolysaccharide producing cultures on the quality of plain set type yogurt. Food Control, 16, 205e209. n, J. J., Na jera, A. F., Guerrero, A. E. C., & Garibay, M. G. (2009). Use of Guzma exopolysaccharide-producing strain of Streptococcus thermophilus in the manufacture of Mexican Panela cheese. Food Science and Technology, 42, 1508e1512. Hassan, A. N. (2008). ADSA Foundation Scholar Award: possibilities and challenges of exopolysaccharide-producing lactic cultures in dairy foods. Journal of Dairy Science, 91, 1282e1298. Hassan, A. N., & Awad, S. (2005). Application of exopolysaccharide producing cultures in reduced fat Cheddar cheese: cryo-scanning electron microscopy observations. Journal of Dairy Science, 88, 4214e4220. IDF (International Dairy Federation). (1982). Cheese and processed cheese. Determination of the total solids content. Nº 4: A. Brussels, Belgium: International Dairy Federation. IDF (International Dairy Federation). (1986). Cheese and processed cheese. Fat content. Nº 5: B. Brussels, Belgium: International Dairy Federation. IDF (International Dairy Federation). (1988). Cheese and processed cheese: Determination of chloride content (potentiometric titration method). Nº 88: A. Brussels, Belgium: International Dairy Federation. IDF (International Dairy Federation). (1993). Milk: Determination of the nitrogen content (Kjeldahl method) and calculation of crude protein content. Nº 20: B. Brussels, Belgium: International Dairy Federation. Irudayaraj, J., Chen, M., & McMahon, D. J. (1999). Texture development in Cheddar cheese during ripening. Canadian Agricultural Engineering, 41, 253e258. Kindstedt, P. S., Zielinski, A., & Almena-Aliste, M. (2001). A post-manufacture method to evaluate the effect of pH on Mozzarella cheese characteristics. The Australian Journal of Dairy Technology, 56, 14e19. Kosikowski, F. (1982). Cheese and fermented milk foods. New York: Elsevier. Lawrence, R. C., Creamer, L. K., & Gilles, J. (1987). Texture development during cheese ripening. Journal of Dairy Science, 70, 1748e1760. Lynch, K. M., McSweeney, P. L. H., Arendt, E. K., Uniacke-Lowe, T., Galle, & Coffey, A. (2014). Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese. International Dairy Journal, 34, 125e134. McSweeney, P. L. H. (2004). Biochemistry of cheese ripening. International Journal of Dairy Technology, 27, 127e144. McSweeney, P. L. H., & Sousa, M. J. (2000). Biochemical pathways for the production of flavour compounds in cheese during ripening: a review. Lait, 80, 293e324. Meilgaard, M., Civille, G. V., & Carr, B. T. (1999). Sensory evaluation techniques (3rd ed.). New York: CRC Press. Merril, R. K., Oberg, C. J., & McMahon, D. J. (1994). A method for manufacturing reduced fat Mozzarella cheese. Journal of Dairy Science, 77, 1783e1789. rio da Agricultura do Brasil. (1997). Regulamento t Ministe ecnico de identidade e qualidade do queijo Prato. Portaria n 358, de 4 de setembro de 1997. Brasília, rio da Agricultura, Pecua ria e Abastecimento (Accessed 03.01.14) Brazil: Ministe http://sistemasweb.agricultura.gov.br/sislegis/action/detalhaAto.do? method¼consultarLegislacaoFederal. Nonogaki, C. O., Monteiro, V. S., & Gigante, M. L. (2007). Metodologia para avaliar a capacidade de derretimento do queijo Prato. Brazilian Journal of Food Technology, 10, 71e77. Oluk, A. C., Güven, M., & Hayaloglu, A. A. (2014). Proteolysis texture and microstructure of low-fat Tulum cheese affected by exopolysaccharide-producing cultures during ripening. International Journal of Food Science and Technology,
R.S.C. Nepomuceno et al. / LWT - Food Science and Technology 72 (2016) 383e389 49, 435e443. Perry, D. B., McMahon, D. J., & Oberg, C. J. (1997). Effect of exopolysaccharideproducing cultures on moisture retention in low fat Mozzarella cheese. Journal of Dairy Science, 80, 799e805. Petersen, B. L., Dave, R. I., McMahon, D. J., Oberg, C. J., & Broadbent, J. R. (2000). Influence of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus on Mozzarella cheese and cheese whey. Journal of Dairy Science, 83, 1952e1956. Rynne, N. M., Beresford, T. P., Kelly, A. L., Tunick, M. H., Malin, E., & Guinee, T. P. (2007). Effect of exopolysaccharide-producing adjunct starter cultures on the manufacture, composition and yield of half-fat Cheddar cheese. Australian Journal Dairy Technology, 62, 12e18. Salaün, F., Mietton, B., & Gaucheron, F. (2005). Buffering capacity of dairy products. International Dairy Journal, 15, 95e109. Sallami, L., Kheadr, E. E., Fliss, I., & Vuillemard, J. C. (2004). Impact of autolytic, proteolytic and nisin-producing adjunct cultures on biochemical and textural properties of Cheddar cheese. Journal of Dairy Science, 87, 1585e1594. S¸anli, T., Gursel, A., S¸anli, E., Acar, E., & Benli, M. (2013). The effect of using an
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exopolysaccharide-producing culture on the physicochemical properties of low-fat and reduced-fat Kasar cheeses. International Journal of Dairy Technology, 66, 535e542. rrez-Me ndez, N., Sepulveda, D. R., & Hern Trancoso-Reyes, N., Gutie andezOchoa, L. R. (2014). Assessing the yield, microstructure, and texture properties of miniature Chihuahua-type cheese manufactured with a phospholipase A1 and exopolysaccharide-producing bacteria. Journal of Dairy Science, 97, 598e608. Walstra, P., Geurts, T. J., Noomen, A., Jellema, A., & Van Boekel, M. A. J. S. (1999). Dairy technology: Principles of milk properties and processes. New York: Marcel Dekker. Zisu, B., & Shah, N. P. (2006). Role of microbial exopolysaccharides on moisture retention and texture and functionality of low fat Mozzarella cheeses. Australian Journal of Dairy Technology, 61, 253e260. Zisu, B., & Shah, N. P. (2007). Texture characteristics and pizza bake properties of low-fat mozzarella cheese as influenced by pre-acidification with citric acid and use of encapsulated and ropy exopolysaccharide producing cultures. International Dairy Journal, 17, 985e997.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Exopolysaccharide-producing culture in the manufacture of Prato cheese Raquel Sant’Ana Coelho Nepomuceno a, Luiz Carlos Gonçalves Costa Junior b, Renata Golin Bueno Costa b, * a
Barbosa & Marques S/A, R. Aluísio Pereira Esteves, 250, 35032-010, Governador Valadares, Minas Gerais, Brazil ria de Minas Gerais, Instituto de Laticínios Ca ^ndido Tostes, R. Tenente Luiz de Freitas, 116, 36045-560, Juiz de Fora, Minas Empresa de Pesquisa Agropecua Gerais, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2015 Received in revised form 25 April 2016 Accepted 26 April 2016 Available online 3 May 2016
Exopolysaccharide (EPS)-producing culture in capsular form has been used in the manufacture of Prato cheese to evaluate the effect on the physicochemical and sensory characteristics, texture properties, melting capacity, and yield of Prato cheese. Prato cheeses were manufactured with and without the addition of EPS-producing culture, and assessed within 120 days of refrigerated storage. The addition of EPS-producing culture increased yield in 0.25 kg/100 kg of milk when compared to the cheese made with EPS non-producing culture. Cheeses manufactured with EPS-producing culture had higher moisture content than the control; however, this did not affect proteolysis, pH, and the melting capacity of cheeses. In addition, cheeses containing EPS had lower hardness and chewiness than the control cheese. Although EPS has changed the texture characteristics, it did not affect the sensory acceptance, which was similar for both treatments. These results indicate that Prato cheeses made with EPS-producing culture have better performance without changing their physicochemical and sensory characteristics. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Prato cheese Brazilian cheese Capsular exopolysaccharide Cheese yield
1. Introduction Exopolysaccharide (EPS)-producing cultures (EPSþ) have been used in cheese making due to the excellent property of EPS to bind water and retain moisture. In low fat cheeses, EPS improves texture and quality, and it becomes similar to its full-fat cheese (Abou Ayana & Ibrahim, 2015; Lynch, McSweeney, Arendt, UniackeLowe, & Coffey, 2014). Encapsulated EPS-producing strains can be used to increase moisture in cheese and improve the melting property of low fat mozzarella, without adversely affecting the whey viscosity, with no changes in cheese properties and processing (De Vuyst et al., 2003; Zisu & Shah, 2007). In reduced fat Cheddar cheese, the use of Lactococcus lactis ssp. cremoris producing EPS in capsular form led to an increase in moisture content and cheese yield, and improved cheese firmness and elasticity after six months of ripening (Dabour, Kheadr, Benhamou, Fliss, & Lapointe, 2006). Previous studies using EPS-producing culture in low fat cheese found an increase in cheese yield due to the higher EPS
* Corresponding author. E-mail address: [email protected] (R.G.B. Costa). http://dx.doi.org/10.1016/j.lwt.2016.04.053 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
water retention capacity (Costa et al., 2010; Di Cagno, De Pasquale, De Angelis, Buchin, Rizzello, & Gobbetti, 2014; Oluk, Güven, & Hayaloglu, 2014; S¸anli, Gursel, S¸anli, Acar, & Benli, 2013). Although several authors have studied low fat cheeses, there are few studies on the effect of EPS-producing cultures in cheese jera, Guerrer and Garibay (2009) without fat reduction. Guzman, Na evaluated the use of EPS-producing Streptococcus thermophilus in the manufacture of Panela Mexican cheese, a soft and moist cheese. These authors have reported higher water retention in cheese, thus rrez-Me ndez, Sepulveda, increasing yield. Trancoso-Reyes, Gutie ndez-Ochoa (2014) also found higher yield and moisand Herna ture and fat retention in Chihuahua cheese made with EPSproducing bacteria. In Brazil, Prato cheese and its varieties correspond about 20% of all cheeses produced in the country (Abiq, 2011). Prato cheese is of Danish origin, similar to Gouda and Danbo, despite its different flavor and texture. It is consumed both directly and indirectly (Cichoscki, Valduga, Valduga, Tornadijo, & Fresno, 2002). Although it has a great representativeness in Brazilian market, there are no studies using EPS-producing culture in the manufacture of Prato cheese. Therefore, the aim of this study was to evaluate the effect of capsular EPS-producing culture on the physicochemical and
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culture used in Prato cheese manufacture was used, consisting of Streptococcus thermophilus, Lactococcus lactis ssp. lactis and/or Lactococcus lactis ssp. cremoris (Lyofast MOS 062 E) (Clerici-Sacco, Caglifio Clerici, Cadorago, Italy), at a dose of 50 UC (77.5 g) for 12,500 L of milk. Analyses of proteolysis, pH, melting capacity, and texture profile were performed after 2, 30, 60, 90, and 120 days of refrigerated storage in all 5 replicates. 2.2. Physicochemical characterization The cheeses were characterized after two days of refrigerated storage in all 5 replicates for total solids, fat levels, protein, and salt content according to IDF International Standards 4A (1982), 5B (1986), 20B (1993) and 88A (1988), respectively. The pH was determined according to AOAC (1980) and total nitrogen (NT) by Kjeldahl method (AOAC, 1995). The pH 4.6- soluble nitrogen and 12% TCA-soluble nitrogen were determined according to Barbano, Lynch, and Fleming (1991), and AOAC (1995), respectively. Proteolysis was evaluated by the extent and depth of proteolysis indexes using the following equations:
Extent of proteolysis ¼ ð% pH 4:6 soluble nitrogen=% total nitrogenÞ 100 (1) Depth of Proteolysis ¼ ð% 12% TCA soluble nitrogen=% total nitrogenÞ 100 (2)
Fig. 1. Flowchart of Prato cheese manufacture.
sensory characteristics, texture properties, melting capacity, and yield of Prato cheese. 2. Material and methods 2.1. Manufacture of Prato cheese Prato cheese was manufactured in 5 replicates on different days. The manufacturing process is described in Fig. 1. Each batch of cheese was made with 12,500 L capacity. Calcium chloride (50% w/ v, 0.30 mL/L milk); annatto dye (0.025 mL/100 L milk - Chr. Hansen Brazil, Valinhos, Brazil), starter culture (UC 50, Unita Cento) and liquid microbial coagulant, 100% chymosin (Clerici-Sacco, Caglifio
The pH of the samples and the proteolysis indexes were determined after 2, 30, 60, 90, and 120 days of refrigerated storage in 5 replicates. Milk was analyzed for fat content by the Gerber method (British Standards Institution, 1989) and total nitrogen (AOAC International, 2006). The protein content was determined by multiplying the total nitrogen content by 6.38. The whey was collected 15 min after cutting the curd, and analyzed for fat and total nitrogen contents according to the methods previously mentioned. Actual yield was expressed as kg cheese/100 kg milk. Fat and protein recoveries were calculated according to Alves, Merheb-Dini, Gomes, Silva, and Gigante (2013). Adjusted yield (Yadj) was calculated considering the desired moisture content of 43% (w/w) and the desired NaCl level of 1.6% (w/w) according to equation (3):
Yadj ¼ fYact ½100 ð%realmoisturecontent þ %realsaltcontentÞg=½100 ð%desirablemoisturecontent þ %desirablesaltcontent
Clerici, Cadorago, Italy) were used to manufacture the cheese. The following treatments were carried out: (1) lyophilized capsular exopolysaccharide-producing culture (EPS culture) for direct inoculation and (2) a traditional culture used in Prato cheese manufacture (control). EPS culture was added to the production tank at a dose of 20 Unita Cento (UC) (49.0 g) for Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris (Lyofast MO 242) and 30 UC (28.5 g) for Streptococcus thermophilus (Lyofast ST 4.40) (ClericiSacco, Caglifio Clerici, Cadorago, Italy). As a control, a traditional
(3)
2.3. Melting capacity of Prato cheese The melting capacity was determined by the modified method of Schreiber (Kosikowski, 1982), in quadruplicate. A cheese cylinder of approximately 36 mm in diameter and 7 mm thick was sampled, and discs were cut with the aid of a slicer. Four slices were sampled from the inner region of the cheeses. Each slice was placed in the
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center of a Petri plate, divided into 8 areas of equal diameters. Four diameters were measured for each sample (Di), and then the plates were placed in an oven at 130 C for 10 min (Nonogaki et al., 2007). These authors studied the best time and temperature condition for maximum melting of Prato cheese, without burning the product. After 10 min, the plates were maintained for 30 min at room temperature and the diameters of each melted sample was measured again (Df). The melting capacity was calculated using the equation:
MCð%Þ ¼
. Df 2 Di2 100 Di2
(4)
where: MC: melting capacity; Df: final diameter; Di: initial diameter. 2.4. Texture profile analysis The texture profile analysis (TPA) of the samples was performed in a CT3 Texture Analyzer (Brookfield, Middleboro, USA) using a 4500 g load cell. The cheeses were cut into cylinders of 20 mm diameter and 25 mm in height, placed in sealed plastic bags (to avoid dehydration) and stored at 10 C for 1 h. Samples were taken from the middle of the cheese block to avoid surface effects. The speed of 1 mm/min was used at 40% compression test, using an acrylic cylindrical probe of 25.4 mm diameter and 35 mm height. 2.5. Sensory evaluation Acceptance test was performed after 30 days of refrigerated storage. Two hundred and fifty untrained assessors (fifty consumers on each sampling day) consisting of students, teachers and staff from Instituto de Laticínios Candido Tostes, of varied ages and both sexes, were selected based on regular consumption of cheese, time availability and interest. The acceptance test was evaluated by a 9-point hedonic scale (1 ¼ extremely disliked, 7 ¼ liked moderately, and 9 ¼ liked extremely; Meilgaard, Civille, & Carr, 1999) for the attributes appearance, flavor, texture, and overall acceptance. Cheese samples were cut into 2.5 2.5 2.5 cm pieces and codified with random 3-digits numbers. 2.6. Statistical analysis Statistical analyses were performed using Minitab 12.1 program
Table 1 Physicochemical composition of Prato cheese made with and without addition of EPS-producing culture after two days of refrigerated storage (means ± SD; n ¼ 5). Composition
Moisture (% w/w) Fat (% w/w) Protein (%w/w) NaCl (%w/w) FDMb (%w/w) Salt in moisture (% w/w) MNFSc Total yield (kg cheese/100 kg milk) Adjusted yieldd (kg cheese/100 kg milk) Fat recovery in cheese Protein recovery in cheese
Culturea
P-value
EPS
EPSþ
41.03 ± 0.94 31.74 ± 1.06 23.40 ± 0.44 0.57 ± 0.24 53.82 ± 0.95 1.37 ± 0.55 60.11 ± 0.49 11.15 ± 0.09 10.37 ± 0.06 92.14 ± 0.42 79.39 ± 1.26
43.86 ± 1.44 29.80 ± 0.45 22.25 ± 1.19 0.87 ± 0.18 53.10 ± 0.65 1.94 ± 0.38 62.47 ± 1.68 11.40 ± 0.20 11.07 ± 0.43 91.64 ± 0.70 78.53 ± 0.68
0.01 0.01 0.07 0.13 0.24 0.18 0.03 0.02 0.03 0.25 0.20
a EPS ¼ cheese manufactured with non-EPS-producing starter; EPSþ ¼ cheese manufactured with EPS-producing starter. b FDM ¼ fat in dry matter. c MNFS ¼ moisture in nonfat substance. d Adjusted yield, considering 43% moisture and 1.6% salt.
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for Windows (Minitab Inc., State College). The effect of culture (2 levels), time of refrigerated storage (5 levels), and their interactions were analyzed by ANOVA and Tukey’s test at a significance level of P < 0.05. 3. Results and discussion 3.1. Physicochemical characterization Both Prato cheeses made with the addition of EPS-producing culture and with conventional procedure (Table 1) met the standards established by Brazilian legislation for moisture content (36% and 45.9% w/w) and fat in the dry matter (45% and 59.9% w/w) rio da Agricultura do Brasil, 1997). (Ministe The use of EPS led to an increase in moisture levels (P < 0.05), due to its water retention capacity. Although the water retention is higher for the EPS-culture in ropy form when compared to the capsular form (Dabour, Kheadr, Fliss, & Lapointe, 2005; Petersen, Dave, McMahon, Oberg, & Broadbent, 2000), a greater water retention was observed in the present study using the capsular form, with an increase in moisture content of 6.89% (w/w). Studies on low fat cheese with the addition of exopolysaccharideproducing culture have also found high moisture levels. In Mozzarella cheese, an increase in moisture content was observed for the samples with EPS-producing cultures in ropy form and capsular form, with values of 6.1% and 2.7%, respectively (Petersen et al., 2000), while a higher water retention of 3.6%e4.8% was observed in low fat Cheddar cheese (Dabour et al., 2006). The increase in moisture levels leads to lower dry extract, with significant changes in cheese fat content, which was lower in the cheeses produced with EPSþ (P < 0.05). However, no significant difference was observed in fat retention between treatments (P > 0.05). This difference in fat content was due the dilution effect of the dry extract constituents (Rynne et al., 2007). Half-fat Cheddar cheese made with EPS-producing strain also had higher moisture and less fat levels than the control (Costa et al., 2010). Moisture in nonfat substance (MNFS) made with EPS-producing culture was higher than the control (P < 0.05). The EPS has the ability to bind water through hydrogen bonds (Costa et al., 2010), increasing MNFS without need to change the cheese making process (Hassan, 2008). Cheeses produced with EPS-producing culture have better actual and adjusted yield than the control cheese (P < 0.05). An increase of 0.25 kg cheese/100 kg of milk in yield of Prato cheese produced with EPSþ was observed, which is extremely important, considering that this calculation is performed to estimate the manufacturing costs of dairy products. The experimental results are consistent with those obtained by Dabour et al. (2006) for reducedfat Cheddar cheese, who reported an increase in yield of 0.28e1.19 kg of cheese/100 kg milk for cheeses produced with EPSproducing strains when compared to the control cheese. However, these values are far below the increase of 1.89 kg per 100 kg milk obtained for the Panela Mexican cheese (Guzman et al., 2009), which has high moisture for being a fresh cheese. In addition, Panela cheese was produced with EPS-producing strain in ropy form, which has higher water retention capacity than the capsular form used in Prato cheese of this study (Dabour et al., 2005; Petersen et al., 2000). The use of EPS-producing bacteria in Chihuahua cheese increased moisture and actual yield (TrancosoReyes et al., 2014). Cheeses made with EPS-producing cultures can present distinct characteristics depending on the variety and nature of the culture (Trancoso-Reyes et al., 2014). No significant differences were observed in fat and protein recovery for all treatments (P > 0.05). The difference in yield was due to the higher moisture levels in cheeses made with EPSþ.
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Table 2 pH and proteolysis indexes of Prato cheese with and without addition of EPS-producing culture for 120 days of refrigerated storage. Cheese
Storage time (days)
pH
Extent of proteolysis index (%)
Depth of proteolysis index (%)
EPSþa
2 30 60 90 120
5.32 5.19 5.23 5.28 5.29
± ± ± ± ±
0.09a 0.16b 0.07b 0.08a 0.03a
6.26 ± 1.10c 6.48 ± 2.91c 7.71 ± 3.55ab 14.69 ± 3.24ab 15.58 ± 3.92a
3.27 ± 0.21b 5.26 ± 0.96b 4.80 ± 1.99b 6.08 ± 1.21b 16.57 ± 7.98a
EPS
2 30 60 90 120
5.32 5.18 5.25 5.31 5.37
± ± ± ± ±
0.09a 0.08b 0.06ab 0.03a 0.05a
7.14 ± 1.97b 8.25 ± 4.67b 8.68 ± 5.15b 13.83 ± 2.00ab 19.97 ± 4.05a
2.47 ± 0.61b 6.81 ± 1.08b 3.73 ± 1.33b 5.52 ± 0.65b 17.19 ± 7.71a
a,b a
Means within the same column and the same treatment with different superscript letters differ significantly (P < 0.05); n ¼ 5. EPSþ ¼ cheese manufactured with EPS producing starter; EPS ¼ cheese manufactured with non-EPS-producing starter.
Fig. 2. Melting capacity of Prato cheese manufactured with EPS-producing culture (EPSþ) and without EPS (EPS) subjected to 130 C/10 min during ripening. abMeans between d 2 to 120 within a cheese type with different letters are significantly different (P < 0.05; n ¼ 5).
3.2. Evolution of pH values and proteolysis indexes No significant changes in pH were observed in Prato cheeses manufactured with EPS-producing culture (P > 0.05). The average
pH values of the cheeses with and without EPS over the 120 days of ripening were 5.28 and 5.32, respectively. In preliminary studies (not shown), the EPS-producing culture was added at the beginning of the manufacture, during tank filling, resulting in rapid decrease of pH, altering the final composition of the cheese. Higher moisture levels in cheeses are due to the greater amount of lactose retained in curd, leading to bacteria growth thus lowering the pH (Walstra, Geurts, Noomen, Jellema, & Van Boekel, 1993). The pH interferes with curd syneresis, besides affecting moisture and enzyme activity during ripening. The reduction of pH affects the rheological properties, melting capacity, and elasticity of cheeses (Fox, 2004, Chap. 1). In cheeses manufactured with EPSþ, there was a reduction in time required to reach pH when compared to the control cheese (Dabour et al., 2006; Dabour et al., 2005). In this study, it was necessary to change the moment of addition of the culture to better control the pH drop during the manufacturing process, so that these values were similar between treatments to avoid interference with the cheese characteristics. Therefore, the culture inoculation was carried out when the tank was filled with milk until at least 80% of its capacity. For non-EPS-producing culture, the inoculation occurred when milk has reached 30% of the tank capacity. At the beginning of ripening, a decrease in pH of the cheese was observed (Table 2), which may have been caused by the fermentation of residual lactose and consequent formation of lactic acid (McSweeney & Sousa, 2000). As expected, there was an increase in pH of the cheeses during ripening due to partial degradation of lactic acid and formation of alkali nitrogenous compounds (Salaün,
Fig. 3. Changes in the rheological properties: hardness (A) and chewiness (B) during ripening of Prato cheese manufactured with (-) and without (B) EPS-producing culture (n ¼ 5).
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Mietton, & Gaucheron, 2005). No significant difference was observed for the extent and depth of proteolysis indexes between treatments (P > 0.05). Although there was an increase in moisture content of the cheese manufactured with EPS-producing culture, no changes were observed in proteolysis. Guzel-Seydim, Sezgin, and Seydim (2005) found that the exopolysaccharide binds strongly to water within the cheese matrix, retaining water for a longer time when compared to control. Even the high moisture content (62.2%) in low-fat mozzarella was stabilized within the cheese matrix (Perry, McMahon, & Oberg, 1997). However, a significant effect of time was observed (P < 0.05) for the extent and depth (Table 2) of proteolysis. The increase in proteolysis during ripening is consistent with other studies (Awad, Hassan, & Muthukumarappan, 2005; Dabour et al., 2006; Sallami, Kheadr, Fliss, & Vuillemard, 2004), due casein degradation by the residual coagulant, forming peptides of high molecular weight and the action of microorganisms in cheese, with greater effects over time (McSweeney, 2004). In the manufacture of Prato cheese, similar lactic acid bacteria concentrations and strains were used, as well as the type and amount of coagulant, differing only in the ability of the bacteria to produce EPS. 3.3. Melting capacity Melting is an important parameter in Prato Cheese, due to its ability to change the original shape when subjected to heat. No significant differences were observed in melting properties of the cheeses made with EPSþ (P > 0.05). During ripening, a significant difference was observed in melting capacity (P < 0.05) (Fig. 2), once proteolysis leads to hydration of the para-casein matrix (Lawrence, Creamer, & Gilles, 1987). In addition, the pH strongly affects moisture-protein interactions and hence the melting of the cheese (Kindstedt, Zielinski, & Almena-Aliste, 2001). Considering that no significant difference was observed in pH between treatments, this parameter did not affect melting capacity. Studies have reported better melting properties in low-fat Mozzarella made with EPS-producing culture due to higher moisture content (Merrill, Oberg, & McMahon, 1994; Zisu & Shah, 2007). However, Zisu and Shah (2006) found no significant difference in melting of cheese produced with capsular EPS-producing culture, which is the same type of EPS used in Prato cheese of this study. 3.4. Texture profile analysis The control cheese (EPS) showed higher firmness when compared to the cheese produced with EPS-producing culture (P < 0.05), as reported by other authors (Awad et al., 2005; Dabour et al., 2006). Cheese produced with EPS-producing culture have more open and porous structure due to the EPS water retention capacity (Costa et al., 2010; Hassan & Awad., 2005) as compared to the cheeses made with non-EPS-producing culture, which have a more dense and compact matrix (Costa et al., 2010). The exopolysaccharide interferes with proteineprotein interactions, reducing cheese firmness (Hassan, 2008) and fills empty spaces in protein matrix (Ayala-Hernandez, Goff, & Corredig, 2008). During ripening, a significant reduction in cheese hardness was observed (P < 0.05) (Fig. 3) due to proteolysis of the protein network by chymosin and hydration of proteins (Zisu & Shah, 2007). There is an interdependence between chewiness and hardness (Irudayaraj, Chen, & McMahon, 1999), and in this study chewiness was lower in cheeses made with EPS-producing culture (P < 0.05). Ahmed, El Soda, Hassan, and Frank (2005) also found lower hardness and chewiness values in Karish cheeses made with EPS-
Fig. 4. Cohesiveness, adhesiveness, and springiness of Prato cheese during ripening: presented data show the values for all cheeses manufactured with and without EPSproducing culture (n ¼ 5).
Table 3 Sensory scores for the cheese manufactured without EPS (control) and with EPSproducing culture after 30 days of refrigerated storage (mean ± SD, n ¼ 5). Attributes
EPSþa
Appearance Flavor Texture Overall acceptance
7.58 7.43 7.33 7.42
± ± ± ±
EPS 1.08 1.37 1.31 1.32
7.54 7.28 7.31 7.17
P-value ± ± ± ±
1.03 1.22 1.29 1.12
0.73 0.28 0.91 0.27
a EPSþ ¼ cheese manufactured with EPS producing starter; EPS ¼ cheese manufactured with non-EPS-producing starter.
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producing culture when compared to those without EPS. No significant differences were observed for the parameters cohesiveness, springiness, and adhesiveness of the cheeses between treatments (P > 0.05). During ripening, a significant effect was observed in all texture characteristics (P < 0.05) (Fig. 4). The texture parameters during ripening are influenced by the proteolytic action of the residual coagulant and lactic acid bacteria (Guinee, 2003; Gunasekaran & Mehmet, 2004). The present results are similar to other studies (Awad et al., 2005; Costa et al., 2010; Dabour et al., 2006). 3.5. Sensory evaluation No significant differences were observed in the acceptance test for all treatments (P > 0.05) for the evaluated parameters (Table 3) after 30 days of refrigerated storage. As the EPS-producing culture did not affect pH and proteolysis, which can interfere with the sensory characteristics of Prato cheese, the untrained assessors did not notice the differences between treatments. Although cheeses containing EPS were less hard and chewy in the texture profile analysis, these characteristics did not affect the sensory acceptance, according to the untrained assessors. 4. Conclusions The use of exopolysaccharide-producing culture in capsular form in the manufacture of Prato cheese improved yield and increased the moisture content. The addition of EPS-producing culture did not affect proteolysis, pH, melting capacity and sensory acceptance. However, differences were observed in the rheological parameters hardness, chewiness. The addition of EPS-producing culture in semi-hard cheeses is a promising alternative to increase yield without changing their physicochemical and sensory characteristics. Acknowledgements The authors thank the financial support to Barbosa & Marques S/ A, Sacco-Brasil, and EPAMIG. References Abou Ayana, I. A. A., & Ibrahim, A. E. (2015). Attributes of low-fat yogurt and Kareish cheese made using exopolysaccharides producing lactic acid bacteria. American Journal of Food Technology, 10, 48e57. Ahmed, N. H., El Soda, M., Hassan, A. N., & Frank, J. (2005). Improving the textural properties of an acid-coagulated (Karish) cheese using exopolysaccharide producing cultures. LWT - Food Science and Technology, 38, 843e847. Alves, L. S., Merheb-Dini, C., Gomes, E., Silva, R. da, & Gigante, M. L. (2013). Yield, changes in proteolysis, and sensory quality of Prato cheese produced with different coagulants. Journal of Dairy Science, 96, 7490e7499. AOAC (Association of Analytical Chemists). (1995). Official methods of analysis of AOAC international. Washington: AOAC. AOAC (Association of Official Analytical Chemists). (1980). Hydrogen-ion activity (pH). In W. Horwitz (Ed.), Official methods of analysis (p. 213). Washington: AOAC. AOAC International. (2006). Official methods of analysis (18th ed.). Arlington, VA: AOAC International. ~o brasileira de pro~o Brasileira das Indústrias de Queijo. (2011). Produça Associaça cteos e estabelecimentos sob inspeça ~o federal. Sa ~o Paulo (N~ dutos la ao paginado). Awad, S., Hassan, A. N., & Muthukumarappan, K. (2005). Application of exopolysaccharide-producing cultures in reduced-fat cheddar cheese: texture and melting properties. Journal of Dairy Science, 88, 4204e4213. Ayala-Hernandez, I., Goff, H. D., & Corredig, M. (2008). Interactions between milk proteins and exopolysaccharides produced by Lactococcus lactis observed by scanning electron microscopy. Journal of Dairy Science, 91, 2583e2590. Barbano, D. M., Lynch, J., & Fleming, J. R. (1991). Direct and indirect determination of true protein content of milk by Kjeldahl analysis: collaborative study. Journal of the AOAC, 74, 281e288. British Standards Institution. (1989). Determination of fat content of milk and milk products (Gerber method)dMethods. London, UK: British Standards Institution. Cichoscki, A. J., Valduga, E., Valduga, A. T., Tornadijo, M. E., & Fresno, J. M. (2002).
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