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Can lutein replace annatto in the manufacture of Prato cheese?
LWT - Food Science and Technology 68 (2016) 349e355
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Can lutein replace annatto in the manufacture of Prato cheese? ~es Machado a, Denise Sobral a, *, Renata Golin Bueno Costa a, Gisela Magalha a ^ Martins Teodoro a, Nata lia Moreira Nunes b, Junio Cesar Jacinto de Paula , Vanessa Aglae Ana Clarissa dos Santos Pires b, Maximiliano Soares Pinto c ^ndido Tostes, Empresa Agropecua ria de Minas Gerais (EPAMIG), Juiz de Fora, MG, 36045-560, Brazil Instituto de Laticínios Ca Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil c rias, Universidade Federal de Minas Gerais, Montes Claros, MG, 39404-006, Brazil Instituto de Ci^ encias Agra a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 October 2015 Received in revised form 14 December 2015 Accepted 23 December 2015 Available online 28 December 2015
Prato cheese is a Brazilian yellow-orange cheese traditionally colored with annatto. However, the aim of this study was to replace annatto with lutein, a natural yellow food pigment with potent antioxidant properties. Accordingly, we evaluated the maintenance of antioxidant activity of two concentrations of lutein (16 and 32 mg L1) in Prato cheese throughout 60 days of ripening, as well as changes in color, pH, texture and ripening profiles, and sensory acceptance of lutein containing Prato cheese. We observed that lutein added to milk was transferred to the curd and antioxidant activity was maintained throughout ripening for both lutein concentrations. The cheese containing the higher concentration of lutein was slightly redder in color than the others; however, the main color changes were due to the ripening. In addition, Prato cheeses containing both lutein concentrations demonstrated similar sensory acceptance compared to cheese containing annatto. Ripening indexes (extension and depth), pH, and texture profile parameters were not affected (p 0.05) by addition of lutein. Therefore, Prato cheese was found to be a good matrix carrier for lutein, maintaining the antioxidant activity of the colorant. Furthermore, lutein can replace annatto without affecting the properties and acceptance of Prato cheese. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Lutein Prato cheese Antioxidant activity Texture Ripening
1. Introduction Age-related macular degeneration (AMD) is a chronic disease of the central part of the retina called the macula, which can lead to loss of vision (Klein et al., 2011; Lim, Mitchell, Seddon, Holz, & Wong, 2012), and can be related to oxidation and inflammation. The effects of AMD can range from the presence of drusen deposits and pigmentary changes in the retina to more serious problems such as geographic atrophy and choroidal neovascularization (Michalska-Małecka, Kabiesz, Nowak, & Spiewak, 2015). In the United States, it is estimated that by 2020, 3 million individuals will have this disease (Klein et al., 2011). Carotenoids can play a role in the human antioxidative system and are generally present in the macula (Michalska-Małecka et al., 2015) as normal macular pigments. Their presence is important once the retina becomes abundantly illuminated and has greater demands for oxygen (Landrum & Bone, 2001). The main macula
* Corresponding author. E-mail address: [email protected] (D. Sobral). http://dx.doi.org/10.1016/j.lwt.2015.12.051 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
carotenoids are lutein, zeaxanthin, and meso-zeaxantin (Landrum & Bone, 2001; Michalska-Małecka et al., 2015). Lutein represents 36% of the carotenoid content of the retina (Landrum & Bone, 2001). Lutein (3,30 -dihydroxy-a-carotene) can be used to fortify food, particularly dairy products that are consumed globally (Najgebauer-lejko, Grega, & Tabaszewska, 2014), and is known to prevent and reduce the occurrence of AMD. As such, recent studies have evaluated the stability of lutein incorporated into dairy products during processing and storage, as well as determine food changes due lutein incorporation (Domingos et al., 2014; Jones, Aryana, & Losso, 2005; Tiaki et al., 2013; Tokusoglu, 2013). Tokusoglu (2013) added different concentrations of lutein in cream cheeses and found that lutein levels did not affect the product flavor. Moreover, this carotenoid remained stable for approximately 6 weeks of storage (Tokusoglu, 2013). Similarly, Domingos et al. (2014) could not identify any differences in yogurt acceptance after adding lutein or riboflavin. In addition, there were no changes in the levels of the compounds levels during storage under light or dark conditions (Domingos et al., 2014). On the other hand, Jones et al. (2005) observed differences in the color of
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cheddar cheese with the addition of lutein compared to cheese without lutein. They also observed differences in microbiological content among cheeses with different concentrations of the carotenoid; however, no lutein degradation was identified (Jones et al., 2005). Prato cheese is a yellow dairy product that is highly consumed in Brazil, similar to Gouda and Danbo cheeses. Traditionally, it is added by annatto (Bixa orellana L.), which contains mainly the bixin pigment; however lutein can also be incorporated as an antioxidant agent into Prato cheese without any negative changes in its color (Sobral et al., 2014; Tiaki et al., 2013). Tiaki et al. (2013) observed high lutein recovery in Prato cheese and thus concluded that the incorporation of lutein in Prato cheese is viable from a technical perspective. However, further studies are needed to understand the lutein antioxidant capacity in Prato cheese. Therefore, this study aimed to evaluate the antioxidant capacity of different concentrations of lutein in Prato cheese during storage. Changes in instrumental colors, texture profile, pH and ripening, and sensory properties were also investigated.
2. Materials and methods 2.1. Materials Commercial lutein and annatto were provided by Royal DSM. Coagulant (Christian Hansen), mesophilic and aromatic cultures (Christian Hansen), as well as bovine raw milk were used in this study.
2.2. Cheese manufacture The Prato cheese was produce in 3 replicates and the cheese manufacturing process is described in Fig. 1. Control cheeses were added by 80 mg L1 of annatto, whereas cheeses containing lutein were added by 16 and 32 mg L1 of the pigment.
2.4. Antioxidant activity Analysis of antioxidant capacity was performed throughout the 60-day ageing period on days 1, 5, 15, 30, and 60 using the ABTS method according to Cai, Luo, Sun, and Corke (2004), with some changes. The ABTSþ radical was obtained through the reaction between ABTS (7.0 mM) and potassium persulfate (2.45 mM; 1:1, v/v) in the absence of light for15 min at 50 C. ABTSþ solution was diluted in ethanol until an absorbance of 0.700 at 734 nm was achieved. Then, 0.5 mL of lutein sample (obtained by extraction as described in section 2.2) was mixed with 3.5 mL of diluted ABTSþ solution, and absorbance at 734 nm was measured. The control and blank samples were prepared by replacing lutein samples with ethanol and extract from cheese without lutein, respectively. Trolox (final concentrations 0e300 mM) was used as a reference standard, and results were expressed as Trolox equivalent antioxidant ability (TE). 2.5. Determination of cheese color Cheese color parameters L, a*, and b* were evaluated according mez-Torres, Garde, Peirote n, and Avila to Go (2015) using a Hunterlab Model Color Quest II colorimeter (Hunter Associates Laboratory, Inc., Reston, VA, USA). Illuminat D65 (standard daylight) with an illumination angle of 10 was used. The method of color measurement was reflectance mode, with specular reflection excluded, were only the diffuse reflectance is measured, which correlates better to the way the observer sees the color of an object. Samples were taken from different points on the pieces of cheese and measured on eight consecutive occasions. Chroma (C*) values were calculated using eq. (1) below.
1=2 C * ¼ a*2 þ b*2
a* and b* values were used to determine Hue angle (H*). If a* and b* values were greater than 0, H* was calculated using eq. (2).
H* ¼ arctg b* a* 2.3. Quantification of lutein Evaluation of lutein concentration in Prato cheese during ripening was carried out according to a previously published protocol (Rodriguez-Amaya, Raymundo, Lee, Simpson, & Hichester, 1976), with minor modifications. Briefly, lutein was extracted from cheese samples (0.20 ± 0.01 g) by sample dilution in 95% ethanol (Vetec) followed by lutein recovery with acetone (Vetec) and petroleum ether (Vetec) in a separation funnel. The samples were then washed with distilled water. The organic solvent was evaporated until dryness using a vacuum rotatory evaporator at 38 C and the lutein resuspended in 5 mL of absolute alcohol. Lutein was quantified by high performance liquid chromatography using a Shimadzu HPLC CLASS-VP with SPD-10AVdetector. The compounds were separated using a reverse phase column C30 YMC (250 4.6 mm, 5 mm) specific to carotenoids, using methanol, tert-butyl-methyl-ether as the mobile phase and tetrahydrofuran as the cleaning solvent, with a flux of 1 mL/min in isocratic mode and injection of 50 mL. Chromatograms were obtained at 450 nm. Samples were resuspended in 5 mL of methanol and filtered using polyethylene membranes with a pore size of 0.22 mm. For quantification, known concentrations of lutein (ranging from 5 to 40 mg L1) were injected in HPLC (>95%) (SigmaeAldrich) for detection. The data from the resulting peaks were used as standards to determine the concentration of lutein samples by performing a regression analysis.
(1)
(2)
If a* < 0 and b* > 0, H* was calculated using eq. (3).
H* ¼ 180 þ arctg b* a*
(3)
But, if a* and b* < 0, H* was calculated using eq. (4).
H* ¼ 270 þ arctg b* a*
(4)
If a* > 0 and b* < 0, H* was calculated using eq. (5).
H* ¼ 360 þ arctg b* a*
(5)
Sensory analyses were performed after 60 days of storage. Cheeses were evaluated using acceptability tests, a structured ninepoint hedonic scale ranging from 1 (extremely dislike) to 9 (like extremely). On each sampling day, 40 consumers from Instituto de ^ndido Tostes (ILCT), including teachers, students, and Lacticínios Ca staff, were recruited based on interests and cheese consuming habits. The middle portions of the aged cheeses (in triplicate) were cut into 2.5 2.5 2.5 cm pieces and codified with three random digits. The sensory evaluation of the present study was approved by the EPAMIG/ILCT Ethics Research Committee and was carried out at the Laboratory of Sensory Analysis of the same institution. 2.6. Analysis of pH and ripening indexes of Prato cheese Samples were analyzed to determine the total nitrogen (TN)
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Fig. 1. Schematic flowchart of Prato cheese manufacture. Annatto was added in control cheese at 80 mg L1 of milk and lutein as added at two different concentrations: 16 and 32 mg L1 of milk.
contents. Non-protein-nitrogen (NPN) content was determined using the trichloroacetic acid technique. True protein content was calculated by subtracting NPN from TN and multiplying by 6.38. The ripening index was measured by the degradation of casein or nitrogen originated from organic material, and was quantified as the ratio (%) of soluble nitrogen at pH 4.6 and TN. This index should increase with advancing maturation. Ripening depth involves all low molecular weight nitrogenous substances formed during processing and was quantified as the ratio of NPN to TN (Pinto et al., 2011). The pH was also measured using a Tec-2 Tecnal pHmeter
at 20-30 C. 2.7. Analysis of the texture profile of Prato cheese Analysis of texture profile of Prato cheese were carried out according to Messens, Van de Walle, Arevalo, Dewettinck, and Huyghebaert (2000) with some modifications. A texturometer Brookfield model CT3 was used for texture profile analysis (TPA). Pre-test, test, and post-test speeds of 1 mm s1 were used, with a compression distance of 40% from the top of the sample. A
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cylindrical probe (TA 11/1000) of 35 mm in diameter was used. A charge cell of 1 kN was moved perpendicularly through cylindrical cheese samples (25 mm in diameter and 25 mm in height), and randomly collected from the whole cheese. The resistance exerted by the samples was automatically registered and firmness, cohesiveness, springiness, chewiness and adhesiveness were calculated using the Brookfield Texture Pro CT software. 2.8. Data analysis The experiments were carried out three times in a block randomized design with a factorial scheme (time lutein concentration). The proteolysis, pH, color, and TPA results at each point in time were evaluated by analysis of variance (ANOVA), using the Tukey test to compare means. The significance level of was set at p < 0.05. The analyses were performed using the MINITAB software, version 14. 3. Results and discussion 3.1. Quantification and antioxidant activity of lutein in Prato cheese The addition of food dye to Prato cheese is important for cheese acceptance, and in the case of lutein there is an additional role associated with its antioxidant property that benefits human health. Since lutein was added before milk coagulation, some amount could be released in whey. Furthermore, lutein could be lost by oxidative mechanisms during cheese ripening. Therefore, determination of the concentration of lutein in cheeses during ripening was conducted (Fig. 2). The majority of the lutein added to milk was transferred to the curd and only a small amount was lost in whey, probably due to lutein hydrophobicity. Tiaki et al. (2013) also observed that around 95% of lutein added to milk was retained in cheese. The concentration of lutein in cheese was mostly constant during ripening, which indicates that there was no oxidative loss of lutein during ripening. Therefore, lutein in Prato cheese is probably protected by
Fig. 2. Media and standard deviation (3 repetitions) of lutein concentration in Prato cheese during the ripening period. Cheese manufactured with milk containing (B) 16 mg L1 and with (-) 32 mg L1.
lipids and proteins and could be carried in this cheese for human consumption. Lutein was reported to be a potent antioxidant that prevents chronic and degenerative diseases (Tan et al., 2016). However, lutein application in food is limited due to its low stability to rrez, exposure to oxygen, light, high temperatures (Matos, Gutie Iglesias, Coca, & Pazos, 2015) and acid conditions (Davidov-Pardo, Gumus, & McClements, 2016). Since the Prato cheese is manufactured in the presence of oxygen and light, involves a heating step, and present a slight acid environment, evaluation of the antioxidant activity of lutein in cheeses during the ripening is very important. Cheeses containing lutein demonstrated high whereas control samples show smaller antioxidant activity, probably related to the primary proteolysis (Gupta, Mann, Kumar, & Sangwan, 2009) and to antioxidants naturally present in Prato cheese. Furthermore, we observed that the higher the concentration of lutein in the cheese, the higher antioxidant activity (p < 0.05). However, there was no difference (p 0.05) in the antioxidant activity in cheeses during ripening, which was constant during all 60 days of ripening (Fig. 3). The stability of the antioxidant activity of lutein in cheese is probably related to the protection of lutein by the protein-lipid network present in cheese. Because of its hydrophobic nature, lutein in plants and animal cells is often found bound to the hydrophobic domains of proteins and lipids (Cai, Huang, & Wang, 2015), and it is known that it is able to interact with milk proteins, such as bovine serum albumin (BSA) (Reszczynska, Welc, Grudzinski, Trebacz, & Gruszecki, 2015). Prato cheese comprises a large hydrophobic region (formed by around 25% lipids and 20% proteins) (Tiaki et al., 2013) that can entrap lutein and protect it against instability factors. According to Cichosz and Czeczot (2004), antioxidants in lipophilic regions are more stable than those in the aqueous phase. Another study demonstrated that annatto presents higher antioxidant activity than lutein (Kiokias & Oreopoulou, 2006). In this work, the antioxidant activity of lutein was greater, probably due to the concentration used to achieve the characteristic color of
Fig. 3. Media and standard deviation (3 repetitions) of antioxidant activity expressed in Trolox equivalent (TE) of lutein present in Prato cheese during ripening period. Cheeses manufactured with milk containing: (:) no lutein, (B) 16 mg L1, and (-) 32 mg L1 of lutein.
D. Sobral et al. / LWT - Food Science and Technology 68 (2016) 349e355
Prato cheese. Kiokias et al. (2006) showed that concentration of carotenoid is more effective in increasing antioxidant activity of lutein than of bixin. Thus, even annatto concentration (used in Prato cheese manufacture) was higher than lutein; there was no increment in the antioxidant activity of the former. Despite of annatto, which is traditionally used in Prato cheese, shows an antioxidant activity, being an effective anticancer agent (Zou & Akoh, 2015), the replacement of annatto by lutein can represent a quite convenient and attractive alternative for supplying lutein intake and prevent age-related macular degeneration (AMD). However, these health benefits provided by lutein could not compromise color and acceptance of Prato cheese. 3.2. Color measurement and sensory analysis Color is an important parameter for cheese acceptance. A yellow-orange color is characteristic of Prato cheese, thus it is important to verify the effect of lutein compared to annatto (B. orellana L.), the natural colorant often used in the manufacture of Prato cheese. The color parameters of cheeses, assessed over60 days of ripening are shown in Fig. 4. The value of L* decreased as ripening increased for all cheeses, thus cheeses became more opaque as ripening followed, probably due to water evaporation during the ripening period. However,
353
there was no difference (p 0.05) in L* values for cheeses with or without lutein, perhaps because the dry matter of the different mez-Torres et al., 2015). cheeses was similar (Go All cheeses showed positive values for a* and b*, indicating reddish and yellowish directions, respectively, which is visually identified as yellow-orange, the characteristic color of Prato cheese (Supplementary Material, S1). Cheese comprising the higher concentration of lutein was found to be slightly more reddish and yellowish than the control sample. This difference in color parameters increased up to day 15 and was relatively constant up until the end of ripening at day 60 (Supplementary Material S2).Cheese is a biochemically and biologically dynamic system that undergoes sequential changes as it goes through the ripening process. Therefore, this result could be attributed to these pronounced changes, mainly the higher water loss at the beginning of the ripening process (Chapeau, Silva, Schuck, Thierry, & Floury, 2016). Other important color parameters are chroma (C*) and hue angle (H*), which indicates color saturation degree and tonality, respectively (Tiaki et al., 2013). C* and H* values are presented in Table 1. For all treatments, C* values increased with time, which can be explained by water evaporation during ripening (Tiaki et al., 2013). Samples containing a higher concentration of lutein (32 mg L1)
Fig. 4. Media and standard deviation (3 repetitions) of lightness (L*) and chromatic coordinates a* and b* of Prato cheeses manufactured with milk containing: (:) no lutein, (B) 16 mg L1, and (-) 32 mg L1 of lutein, during ripening.
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Table 1 Chroma (C*) and hue angle (H*) values for Prato cheese during ripening time. Lutein concentration in milk (mg.L1)
C*
H*
Days
0 16 32
1
15
30
60
1
15
30
60
28.09 33.21 38.56
30.81 35.80 43.96
36.85 41.19 48.83
37.81 41.89 49.52
67.83 111.65 79.76
64.26 94.76 70.65
57.24 77.49 67.22
57.33 83.91 66.65
demonstrated similar values to the control treatment. The addition of lutein also changed the tonality of Prato cheeses; however, the H* values of control and cheese manufactured with a higher concentration of lutein were similar. The H* values indicate that control and higher lutein cheeses were orange, while cheese comprising low concentrations of lutein were yellow. Fig. 5 illustrates the difference between C* and H* in cheeses at day 60 of ripening. To confirm whether the difference in instrumental measurements of color due to the addition of lutein affected the acceptance of Prato cheeses, the global acceptance was evaluated by sensory analysis. Sensory evaluation was carried out by 40 non-trained individuals (23 women and 17 men between 20 and 43 years old) using a9-point scale on the last day of ripening (day 60). The average scores of global acceptance of Prato cheeses did not change (p 0.05) with replacement of annatto with lutein, and were 7.40, 6.95, and 7.35 for samples without lutein, and with 16 and32 mg L1 of lutein, respectively. These results corroborate the proximity in the color obtained by instrumental measurements, and indicate that lutein can be used as a natural pigment for Prato cheese. In addition, it is important to emphasize that even lutein presents higher cost than annatto; a small amount of this pigment (less than half concentration) was able to get desirable color of Prato cheese to consumers. Also, cheese containing lutein showed an additional advance related to its protection against AMD. 3.3. Ripening index, pH, and texture profile analysis Prato cheese is a ripened cheese often consumed after 30 days of ripening (Chaves & Gigante, 2016). Therefore, proteolysis is an important characteristic of this cheese. Ripening indices such as extension and depth of ripening were determined for cheeses with or without lutein since they involve the degradation of casein mainly by coagulant and low molecular weight nitrogenous
substances formed during ripening, respectively (Merheb-Dini, Garcia, Penna, Gomes, & da Silva, 2012). Ripening time influenced both indices (extension and depth of ripening) (p < 0.05) (Supplementary Material S3); however, there was no difference (p 0.05) in these parameters caused by lutein, indicating that this pigment did not change the ripening profile of Prato cheese. The pH is another important parameter of cheeses because it indicates changes in the ripening process caused by lactic acid bacteria. It depends on calcium phosphate and protein contents, salting conditions, and the salt sensitivity of the starter culture (Hou, Hannon, McSweeney, Beresford, & Guinee, 2012). For all treatments, the pH increased during ripening (p < 0.05), indicating proteolysis of the proteinous net of the cheese (Voigt et al., 2012). However, there was no difference (p 0.05) between the control or lutein-enriched cheese samples, and the average pH ranged from 5.2 to 5.5, as reported in other studies (Cichoscki, Valduga, Valduga, Tornadijo, & Fresno, 2002; Merheb-Dini et al., 2012). Therefore, replacement of annatto with lutein did not affect the pH increase during cheese ripening. To complete the characterization of Prato cheese, the texture profile of cheeses was evaluated during ripening. The texture profile is a very important parameter for characterizing cheeses as well as for consumer acceptance (Medeiros, Kongo, Borges, & Ponte, 2014). Firmness, springiness, and chewiness parameters decreased (p < 0.05) during ripening. These results can be explained by proteolysis of the casein network initially caused by the residual action of coagulants and then by proteolytic enzymes produced by lactic acid bacteria (LAB), which lead to a softer and lez et al., 2015). less elastic cheese (Ve The addition of lutein did not change (p 0.05) any texture profile parameters of the cheeses, thus the addition of lutein did not affect the ripening of Prato cheese (Table 3, in Supplementary Material S4).
4. Conclusion
Fig. 5. Graphical representation of C* and H* values for cheeses manufactured with milk containing: (A) 32 mg L1, (B) 16 mg L1 of lutein and (C) no lutein, at day 60 of ripening.
For the first time, the antioxidant activity of lutein carried by Prato cheese was evaluated during the 60 days of ripening. The cheese matrix formed by proteins and lipids protected the lutein during the ripening process, since there was no loss of lutein or its antioxidant capacity. Prato cheese with the highest concentration of lutein showed similar coloration (yellow-orange color) to traditional Prato cheeses produced with annatto, and the replacement of annatto with lutein did not influence the sensory acceptance of the cheese. The addition of lutein did not influence the maturation index, pH, and cheese texture profile compared to control during ripening. Prato cheese has significant potential as a lutein carrier, while maintaining the functional properties of this antioxidant. Furthermore, lutein was found to be innocuous to the cheese and may be used as a substitute for the annatto inducing functional properties of Prato cheeses.
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Acknowledgments ~o de Amparo The authors thank Fundaça a Pesquisa do estado de Minas Gerias (FAPEMIG) and Conselho Nacional de Deenvolvigico (CNPq) for the financial support. mento Científico e Tecnolo N.M.N thanks CNPq for the scholarship. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.lwt.2015.12.051. References Cai, X., Huang, Q., & Wang, S. (2015). Isolation of a novel lutein-protein complex from Chlorella vulgaris and its functional properties. Food & Function, 6(6), 1893e1899. http://doi.org/10.1039/c4fo01096e. Cai, Y., Luo, Q., Sun, M., & Corke, H. (2004). Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer (vol. 74, pp. 2157e2184). http://doi.org/10.1016/j.lfs.2003.09.047. Chapeau, A. L., Silva, J. V. C., Schuck, P., Thierry, A., & Floury, J. (2016). The influence of cheese composition and microstructure on the diffusion of macromolecules: a study using fluorescence recovery after photobleaching (FRAP). Food Chemistry, 192, 660e667. http://doi.org/10.1016/j.foodchem.2015.07.053. Chaves, K. S., & Gigante, M. L. (2016). Prato cheese as suitable carrier for Lactobacillus acidophilus La5 and Bifidobacterium Bb12. International Dairy Journal, 52, 10e18. http://doi.org/10.1016/j.idairyj.2015.08.009. Cichoscki, A. J., Valduga, E., Valduga, A. T., Tornadijo, M. E., & Fresno, J. M. (2002). Characterization of Prato cheese, a Brazilian semi-hard cow variety: evolution of physico-chemical parameters and mineral composition during ripening. Food Control, 13(4e5), 329e336. http://doi.org/10.1016/S0956-7135(02)00039-7. Cichosz, G., & Czeczot, H. G. M. (2004). Wartos c biologiczna mleka e ocena poprzez pomiar całkowitego statusu antyoksydacyjnego. Przegl. Mlecz, 2, 4e8. Davidov-Pardo, G., Gumus, C. E., & McClements, D. J. (2016). Lutein-enriched emulsion-based delivery systems: Influence of pH and temperature on physical and chemical stability. Food Chemistry, 196, 821e827. Domingos, L. D., Xavier, a a O., Mercadante, a Z., Petenate, a J., Jorge, R. a, & Viotto, W. H. (2014). Oxidative stability of yogurt with added lutein dye. Journal of Dairy Science, 97(2), 616e623. http://doi.org/10.3168/jds.2013-6971. & Avila, mez-Torres, N., Garde, S., Peirote n, A., Go M. (2015). Impact of Clostridium spp. on cheese characteristics: microbiology, color, formation of volatile compounds and off-flavors. Food Control, 56, 186e194. http://doi.org/10.1016/j. foodcont.2015.03.025. Gupta, A., Mann, B., Kumar, R., & Sangwan, R. B. (2009). Antioxidant activity of cheddar cheeses at differents stages of ripening. International Journal of Dairy Technology, 62, 339e347. http://doi.org/10.1111/j.1471-0307. Hou, J., Hannon, J. A., McSweeney, P. L. H., Beresford, T. P., & Guinee, T. P. (2012). Effect of curd washing on composition, lactose metabolism, pH, and the growth of non-starter lactic acid bacteria in full-fat cheddar cheese. International Dairy Journal, 25(1), 21e28. http://doi.org/10.1016/j.idairyj.2011.12.015. Jones, S. T., Aryana, K. J., & Losso, J. N. (2005). Storage stability of lutein during ripening of cheddar cheese. Journal of Dairy Science, 88(5), 1661e1670. http:// doi.org/10.3168/jds.S0022-0302(05)72838-1. Kiokias, S., & Oreopoulou, V. (2006). Antioxidant properties of natural carotenoid extracts against the AAPH-initiated oxidation of food emulsions. Innovative Food Science and Emerging Technologies, 7(1e2), 132e139. http://doi.org/10.1016/j. ifset.2005.12.004. Klein, R., Chou, C., Klein, B. E. K., Zhang, X., Meuer, S. M., & Saaddine, J. B. (2011). Prevalence of age-related macular degeneration in the US Population (vol. 129 (1), pp. 75e80).
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Landrum, J. T., & Bone, R. A. (2001). Minireview lutein, zeaxanthin, and the macular pigment (vol. 385(1), pp. 28e40). http://doi.org/10.1006/abbi.2000.2171. Lim, L. S., Mitchell, P., Seddon, J. M., Holz, F. G., & Wong, T. Y. (2012). Age-related macular degeneration. Lancet, 379(9827), 1728e1738. http://doi.org/10.1016/ S0140-6736(12)60282-7. rrez, G., Iglesias, O., Coca, J., & Pazos, C. (2015). Characterization, Matos, M., Gutie stability and rheology of highly concentrated monodisperse emulsions containing lutein. Food Hydrocolloids, 49, 156e163. http://doi.org/10.1016/j.foodhyd. 2015.03.021. Medeiros, V., Kongo, J. M., Borges, A., & Ponte, D. J. B. (2014). A study of the physicochemical, electrophoretic and rheological properties throught ripening of S~ ao Jorge (PDO) raw Cow's milk cheese. Journal of Food Technology, 3, 66e72. Merheb-Dini, C., Garcia, G. A. C., Penna, A. L. B., Gomes, E., & da Silva, R. (2012). Use of a new milk-clotting protease from thermomucor indicae-seudaticae N31 as coagulant and changes during ripening of Prato cheese. Food Chemistry, 130(4), 859e865. http://doi.org/10.1016/j.foodchem.2011.07.105. Messens, W., Van de Walle, D., Arevalo, J., Dewettinck, K., & Huyghebaert, A. (2000). Rheological properties of high-pressure-treated gouda cheese. Michalska-Małecka, K., Kabiesz, a., Nowak, M., & Spiewak, D. (2015). Age related macular degeneration e challenge for future: pathogenesis and new perspectives for the treatment. European Geriatric Medicine, 6(1), 69e75. http://doi.org/ 10.1016/j.eurger.2014.09.007. Najgebauer-lejko, D., Grega, T., & Tabaszewska, M. (2014). Yoghurts with addition of selected vegetables: acidity, antioxidant properties and sensory quality. Acta Scientiarum Polonorum - Technologia Alimentaria, 13(1), 35e42. Pinto, M. S., de Carvalho, A. F., Pires, A. C., dos, S., Campos Souza, A. A., Fonseca da Silva, P. H., et al. (2011). The effects of nisin on Staphylococcus aureus count and the physicochemical properties of traditional minas serro cheese. International Dairy Journal, 21(2), 90e96. http://doi.org/10.1016/j.idairyj.2010.08.001. Reszczynska, E., Welc, R., Grudzinski, W., Trebacz, K., & Gruszecki, W. I. (2015). Carotenoid binding to proteins: Modeling pigment transport to lipid membranes. Archives of Biochemistry and Biophysics, 584, 125e133 (September) http://doi.org/10.1016/j.abb.2015.09.004. Rodriguez-Amaya, D. B., Raymundo, L. C., Lee, T., Simpson, K. L., & Hichester, C. O. (1976). Carotenoid pigment changes in ripening Momordica charantia fruits. Annals of Botany, 40, 615e624. Sobral, D., Costa, R. G. B., Teodoro, V. A. M., Moreira, G. D. M. M., Paula, J. C. J. De, & Novaes, T. O. (2014). Quantificaç~ ao e porcentagem de recuperaç~ ao de luteína ~o de queijo prato esfe rico. Revista Do Instituto de Latadicionada na fabricaça ^ndido Tostes, 69(6), 405. http://doi.org/10.14295/2238-6416.v69i6.346. icínios Ca Tan, T. B., Yussof, N. S., Abas, F., Mirhosseini, H., Nehdi, I. A., & Tan, C. P. (2016). Forming a lutein nanodispersion via solvent displacement method: the effects of processing parameters and emulsifiers with different stabilizing mechanisms. Food Chemistry, 194, 416e423. http://doi.org/10.1016/j.foodchem.2015. 08.045. Tiaki, M., Kubo, K., Maus, D., Augusta, A., Xavier, O., Mercadante, A. Z., et al. (2013). Transference of lutein during cheese making, color stability, and sensory acceptance of Prato cheese. Ci^ encia E Tecnologia de Alimentos, 33, 81e88. Tokusoglu, O. (2013). The physicochemical, microbiological, organoleptic properties and antioxidant activities of functional cream cheeses fortified with lutein (vol. 66(4), pp. 527e534). http://doi.org/10.1111/1471-0307.12070. lez, M. A., Bergamini, C. V., Ramonda, M. B., Candioti, M. C., Hynes, E. R., & Ve Perotti, M. C. (2015). Influence of cheese making technologies on plasmin and coagulant associated proteolysis. LWT e Food Science and Technology, 64(1), 282e288. http://doi.org/10.1016/j.lwt.2015.05.053. Voigt, D. D., Chevalier, F., Donaghy, J. A., Patterson, M. F., Qian, M. C., & Kelly, A. L. (2012). Effect of high-pressure treatment of milk for cheese manufacture on proteolysis, lipolysis, texture and functionality of cheddar cheese during ripening. Innovative Food Science & Emerging Technologies, 13, 23e30. http://doi. org/10.1016/j.ifset.2011.10.004. Zou, L., & Akoh, C. C. (2015). Antioxidant activities of annatto and palm tocotrienolrich fractions in fish oil and structured lipid-based infant formula emulsion. Food Chemistry, 168, 504e511.
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Can lutein replace annatto in the manufacture of Prato cheese? ~es Machado a, Denise Sobral a, *, Renata Golin Bueno Costa a, Gisela Magalha a ^ Martins Teodoro a, Nata lia Moreira Nunes b, Junio Cesar Jacinto de Paula , Vanessa Aglae Ana Clarissa dos Santos Pires b, Maximiliano Soares Pinto c ^ndido Tostes, Empresa Agropecua ria de Minas Gerais (EPAMIG), Juiz de Fora, MG, 36045-560, Brazil Instituto de Laticínios Ca Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil c rias, Universidade Federal de Minas Gerais, Montes Claros, MG, 39404-006, Brazil Instituto de Ci^ encias Agra a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 October 2015 Received in revised form 14 December 2015 Accepted 23 December 2015 Available online 28 December 2015
Prato cheese is a Brazilian yellow-orange cheese traditionally colored with annatto. However, the aim of this study was to replace annatto with lutein, a natural yellow food pigment with potent antioxidant properties. Accordingly, we evaluated the maintenance of antioxidant activity of two concentrations of lutein (16 and 32 mg L1) in Prato cheese throughout 60 days of ripening, as well as changes in color, pH, texture and ripening profiles, and sensory acceptance of lutein containing Prato cheese. We observed that lutein added to milk was transferred to the curd and antioxidant activity was maintained throughout ripening for both lutein concentrations. The cheese containing the higher concentration of lutein was slightly redder in color than the others; however, the main color changes were due to the ripening. In addition, Prato cheeses containing both lutein concentrations demonstrated similar sensory acceptance compared to cheese containing annatto. Ripening indexes (extension and depth), pH, and texture profile parameters were not affected (p 0.05) by addition of lutein. Therefore, Prato cheese was found to be a good matrix carrier for lutein, maintaining the antioxidant activity of the colorant. Furthermore, lutein can replace annatto without affecting the properties and acceptance of Prato cheese. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Lutein Prato cheese Antioxidant activity Texture Ripening
1. Introduction Age-related macular degeneration (AMD) is a chronic disease of the central part of the retina called the macula, which can lead to loss of vision (Klein et al., 2011; Lim, Mitchell, Seddon, Holz, & Wong, 2012), and can be related to oxidation and inflammation. The effects of AMD can range from the presence of drusen deposits and pigmentary changes in the retina to more serious problems such as geographic atrophy and choroidal neovascularization (Michalska-Małecka, Kabiesz, Nowak, & Spiewak, 2015). In the United States, it is estimated that by 2020, 3 million individuals will have this disease (Klein et al., 2011). Carotenoids can play a role in the human antioxidative system and are generally present in the macula (Michalska-Małecka et al., 2015) as normal macular pigments. Their presence is important once the retina becomes abundantly illuminated and has greater demands for oxygen (Landrum & Bone, 2001). The main macula
* Corresponding author. E-mail address: [email protected] (D. Sobral). http://dx.doi.org/10.1016/j.lwt.2015.12.051 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
carotenoids are lutein, zeaxanthin, and meso-zeaxantin (Landrum & Bone, 2001; Michalska-Małecka et al., 2015). Lutein represents 36% of the carotenoid content of the retina (Landrum & Bone, 2001). Lutein (3,30 -dihydroxy-a-carotene) can be used to fortify food, particularly dairy products that are consumed globally (Najgebauer-lejko, Grega, & Tabaszewska, 2014), and is known to prevent and reduce the occurrence of AMD. As such, recent studies have evaluated the stability of lutein incorporated into dairy products during processing and storage, as well as determine food changes due lutein incorporation (Domingos et al., 2014; Jones, Aryana, & Losso, 2005; Tiaki et al., 2013; Tokusoglu, 2013). Tokusoglu (2013) added different concentrations of lutein in cream cheeses and found that lutein levels did not affect the product flavor. Moreover, this carotenoid remained stable for approximately 6 weeks of storage (Tokusoglu, 2013). Similarly, Domingos et al. (2014) could not identify any differences in yogurt acceptance after adding lutein or riboflavin. In addition, there were no changes in the levels of the compounds levels during storage under light or dark conditions (Domingos et al., 2014). On the other hand, Jones et al. (2005) observed differences in the color of
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cheddar cheese with the addition of lutein compared to cheese without lutein. They also observed differences in microbiological content among cheeses with different concentrations of the carotenoid; however, no lutein degradation was identified (Jones et al., 2005). Prato cheese is a yellow dairy product that is highly consumed in Brazil, similar to Gouda and Danbo cheeses. Traditionally, it is added by annatto (Bixa orellana L.), which contains mainly the bixin pigment; however lutein can also be incorporated as an antioxidant agent into Prato cheese without any negative changes in its color (Sobral et al., 2014; Tiaki et al., 2013). Tiaki et al. (2013) observed high lutein recovery in Prato cheese and thus concluded that the incorporation of lutein in Prato cheese is viable from a technical perspective. However, further studies are needed to understand the lutein antioxidant capacity in Prato cheese. Therefore, this study aimed to evaluate the antioxidant capacity of different concentrations of lutein in Prato cheese during storage. Changes in instrumental colors, texture profile, pH and ripening, and sensory properties were also investigated.
2. Materials and methods 2.1. Materials Commercial lutein and annatto were provided by Royal DSM. Coagulant (Christian Hansen), mesophilic and aromatic cultures (Christian Hansen), as well as bovine raw milk were used in this study.
2.2. Cheese manufacture The Prato cheese was produce in 3 replicates and the cheese manufacturing process is described in Fig. 1. Control cheeses were added by 80 mg L1 of annatto, whereas cheeses containing lutein were added by 16 and 32 mg L1 of the pigment.
2.4. Antioxidant activity Analysis of antioxidant capacity was performed throughout the 60-day ageing period on days 1, 5, 15, 30, and 60 using the ABTS method according to Cai, Luo, Sun, and Corke (2004), with some changes. The ABTSþ radical was obtained through the reaction between ABTS (7.0 mM) and potassium persulfate (2.45 mM; 1:1, v/v) in the absence of light for15 min at 50 C. ABTSþ solution was diluted in ethanol until an absorbance of 0.700 at 734 nm was achieved. Then, 0.5 mL of lutein sample (obtained by extraction as described in section 2.2) was mixed with 3.5 mL of diluted ABTSþ solution, and absorbance at 734 nm was measured. The control and blank samples were prepared by replacing lutein samples with ethanol and extract from cheese without lutein, respectively. Trolox (final concentrations 0e300 mM) was used as a reference standard, and results were expressed as Trolox equivalent antioxidant ability (TE). 2.5. Determination of cheese color Cheese color parameters L, a*, and b* were evaluated according mez-Torres, Garde, Peirote n, and Avila to Go (2015) using a Hunterlab Model Color Quest II colorimeter (Hunter Associates Laboratory, Inc., Reston, VA, USA). Illuminat D65 (standard daylight) with an illumination angle of 10 was used. The method of color measurement was reflectance mode, with specular reflection excluded, were only the diffuse reflectance is measured, which correlates better to the way the observer sees the color of an object. Samples were taken from different points on the pieces of cheese and measured on eight consecutive occasions. Chroma (C*) values were calculated using eq. (1) below.
1=2 C * ¼ a*2 þ b*2
a* and b* values were used to determine Hue angle (H*). If a* and b* values were greater than 0, H* was calculated using eq. (2).
H* ¼ arctg b* a* 2.3. Quantification of lutein Evaluation of lutein concentration in Prato cheese during ripening was carried out according to a previously published protocol (Rodriguez-Amaya, Raymundo, Lee, Simpson, & Hichester, 1976), with minor modifications. Briefly, lutein was extracted from cheese samples (0.20 ± 0.01 g) by sample dilution in 95% ethanol (Vetec) followed by lutein recovery with acetone (Vetec) and petroleum ether (Vetec) in a separation funnel. The samples were then washed with distilled water. The organic solvent was evaporated until dryness using a vacuum rotatory evaporator at 38 C and the lutein resuspended in 5 mL of absolute alcohol. Lutein was quantified by high performance liquid chromatography using a Shimadzu HPLC CLASS-VP with SPD-10AVdetector. The compounds were separated using a reverse phase column C30 YMC (250 4.6 mm, 5 mm) specific to carotenoids, using methanol, tert-butyl-methyl-ether as the mobile phase and tetrahydrofuran as the cleaning solvent, with a flux of 1 mL/min in isocratic mode and injection of 50 mL. Chromatograms were obtained at 450 nm. Samples were resuspended in 5 mL of methanol and filtered using polyethylene membranes with a pore size of 0.22 mm. For quantification, known concentrations of lutein (ranging from 5 to 40 mg L1) were injected in HPLC (>95%) (SigmaeAldrich) for detection. The data from the resulting peaks were used as standards to determine the concentration of lutein samples by performing a regression analysis.
(1)
(2)
If a* < 0 and b* > 0, H* was calculated using eq. (3).
H* ¼ 180 þ arctg b* a*
(3)
But, if a* and b* < 0, H* was calculated using eq. (4).
H* ¼ 270 þ arctg b* a*
(4)
If a* > 0 and b* < 0, H* was calculated using eq. (5).
H* ¼ 360 þ arctg b* a*
(5)
Sensory analyses were performed after 60 days of storage. Cheeses were evaluated using acceptability tests, a structured ninepoint hedonic scale ranging from 1 (extremely dislike) to 9 (like extremely). On each sampling day, 40 consumers from Instituto de ^ndido Tostes (ILCT), including teachers, students, and Lacticínios Ca staff, were recruited based on interests and cheese consuming habits. The middle portions of the aged cheeses (in triplicate) were cut into 2.5 2.5 2.5 cm pieces and codified with three random digits. The sensory evaluation of the present study was approved by the EPAMIG/ILCT Ethics Research Committee and was carried out at the Laboratory of Sensory Analysis of the same institution. 2.6. Analysis of pH and ripening indexes of Prato cheese Samples were analyzed to determine the total nitrogen (TN)
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Fig. 1. Schematic flowchart of Prato cheese manufacture. Annatto was added in control cheese at 80 mg L1 of milk and lutein as added at two different concentrations: 16 and 32 mg L1 of milk.
contents. Non-protein-nitrogen (NPN) content was determined using the trichloroacetic acid technique. True protein content was calculated by subtracting NPN from TN and multiplying by 6.38. The ripening index was measured by the degradation of casein or nitrogen originated from organic material, and was quantified as the ratio (%) of soluble nitrogen at pH 4.6 and TN. This index should increase with advancing maturation. Ripening depth involves all low molecular weight nitrogenous substances formed during processing and was quantified as the ratio of NPN to TN (Pinto et al., 2011). The pH was also measured using a Tec-2 Tecnal pHmeter
at 20-30 C. 2.7. Analysis of the texture profile of Prato cheese Analysis of texture profile of Prato cheese were carried out according to Messens, Van de Walle, Arevalo, Dewettinck, and Huyghebaert (2000) with some modifications. A texturometer Brookfield model CT3 was used for texture profile analysis (TPA). Pre-test, test, and post-test speeds of 1 mm s1 were used, with a compression distance of 40% from the top of the sample. A
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cylindrical probe (TA 11/1000) of 35 mm in diameter was used. A charge cell of 1 kN was moved perpendicularly through cylindrical cheese samples (25 mm in diameter and 25 mm in height), and randomly collected from the whole cheese. The resistance exerted by the samples was automatically registered and firmness, cohesiveness, springiness, chewiness and adhesiveness were calculated using the Brookfield Texture Pro CT software. 2.8. Data analysis The experiments were carried out three times in a block randomized design with a factorial scheme (time lutein concentration). The proteolysis, pH, color, and TPA results at each point in time were evaluated by analysis of variance (ANOVA), using the Tukey test to compare means. The significance level of was set at p < 0.05. The analyses were performed using the MINITAB software, version 14. 3. Results and discussion 3.1. Quantification and antioxidant activity of lutein in Prato cheese The addition of food dye to Prato cheese is important for cheese acceptance, and in the case of lutein there is an additional role associated with its antioxidant property that benefits human health. Since lutein was added before milk coagulation, some amount could be released in whey. Furthermore, lutein could be lost by oxidative mechanisms during cheese ripening. Therefore, determination of the concentration of lutein in cheeses during ripening was conducted (Fig. 2). The majority of the lutein added to milk was transferred to the curd and only a small amount was lost in whey, probably due to lutein hydrophobicity. Tiaki et al. (2013) also observed that around 95% of lutein added to milk was retained in cheese. The concentration of lutein in cheese was mostly constant during ripening, which indicates that there was no oxidative loss of lutein during ripening. Therefore, lutein in Prato cheese is probably protected by
Fig. 2. Media and standard deviation (3 repetitions) of lutein concentration in Prato cheese during the ripening period. Cheese manufactured with milk containing (B) 16 mg L1 and with (-) 32 mg L1.
lipids and proteins and could be carried in this cheese for human consumption. Lutein was reported to be a potent antioxidant that prevents chronic and degenerative diseases (Tan et al., 2016). However, lutein application in food is limited due to its low stability to rrez, exposure to oxygen, light, high temperatures (Matos, Gutie Iglesias, Coca, & Pazos, 2015) and acid conditions (Davidov-Pardo, Gumus, & McClements, 2016). Since the Prato cheese is manufactured in the presence of oxygen and light, involves a heating step, and present a slight acid environment, evaluation of the antioxidant activity of lutein in cheeses during the ripening is very important. Cheeses containing lutein demonstrated high whereas control samples show smaller antioxidant activity, probably related to the primary proteolysis (Gupta, Mann, Kumar, & Sangwan, 2009) and to antioxidants naturally present in Prato cheese. Furthermore, we observed that the higher the concentration of lutein in the cheese, the higher antioxidant activity (p < 0.05). However, there was no difference (p 0.05) in the antioxidant activity in cheeses during ripening, which was constant during all 60 days of ripening (Fig. 3). The stability of the antioxidant activity of lutein in cheese is probably related to the protection of lutein by the protein-lipid network present in cheese. Because of its hydrophobic nature, lutein in plants and animal cells is often found bound to the hydrophobic domains of proteins and lipids (Cai, Huang, & Wang, 2015), and it is known that it is able to interact with milk proteins, such as bovine serum albumin (BSA) (Reszczynska, Welc, Grudzinski, Trebacz, & Gruszecki, 2015). Prato cheese comprises a large hydrophobic region (formed by around 25% lipids and 20% proteins) (Tiaki et al., 2013) that can entrap lutein and protect it against instability factors. According to Cichosz and Czeczot (2004), antioxidants in lipophilic regions are more stable than those in the aqueous phase. Another study demonstrated that annatto presents higher antioxidant activity than lutein (Kiokias & Oreopoulou, 2006). In this work, the antioxidant activity of lutein was greater, probably due to the concentration used to achieve the characteristic color of
Fig. 3. Media and standard deviation (3 repetitions) of antioxidant activity expressed in Trolox equivalent (TE) of lutein present in Prato cheese during ripening period. Cheeses manufactured with milk containing: (:) no lutein, (B) 16 mg L1, and (-) 32 mg L1 of lutein.
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Prato cheese. Kiokias et al. (2006) showed that concentration of carotenoid is more effective in increasing antioxidant activity of lutein than of bixin. Thus, even annatto concentration (used in Prato cheese manufacture) was higher than lutein; there was no increment in the antioxidant activity of the former. Despite of annatto, which is traditionally used in Prato cheese, shows an antioxidant activity, being an effective anticancer agent (Zou & Akoh, 2015), the replacement of annatto by lutein can represent a quite convenient and attractive alternative for supplying lutein intake and prevent age-related macular degeneration (AMD). However, these health benefits provided by lutein could not compromise color and acceptance of Prato cheese. 3.2. Color measurement and sensory analysis Color is an important parameter for cheese acceptance. A yellow-orange color is characteristic of Prato cheese, thus it is important to verify the effect of lutein compared to annatto (B. orellana L.), the natural colorant often used in the manufacture of Prato cheese. The color parameters of cheeses, assessed over60 days of ripening are shown in Fig. 4. The value of L* decreased as ripening increased for all cheeses, thus cheeses became more opaque as ripening followed, probably due to water evaporation during the ripening period. However,
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there was no difference (p 0.05) in L* values for cheeses with or without lutein, perhaps because the dry matter of the different mez-Torres et al., 2015). cheeses was similar (Go All cheeses showed positive values for a* and b*, indicating reddish and yellowish directions, respectively, which is visually identified as yellow-orange, the characteristic color of Prato cheese (Supplementary Material, S1). Cheese comprising the higher concentration of lutein was found to be slightly more reddish and yellowish than the control sample. This difference in color parameters increased up to day 15 and was relatively constant up until the end of ripening at day 60 (Supplementary Material S2).Cheese is a biochemically and biologically dynamic system that undergoes sequential changes as it goes through the ripening process. Therefore, this result could be attributed to these pronounced changes, mainly the higher water loss at the beginning of the ripening process (Chapeau, Silva, Schuck, Thierry, & Floury, 2016). Other important color parameters are chroma (C*) and hue angle (H*), which indicates color saturation degree and tonality, respectively (Tiaki et al., 2013). C* and H* values are presented in Table 1. For all treatments, C* values increased with time, which can be explained by water evaporation during ripening (Tiaki et al., 2013). Samples containing a higher concentration of lutein (32 mg L1)
Fig. 4. Media and standard deviation (3 repetitions) of lightness (L*) and chromatic coordinates a* and b* of Prato cheeses manufactured with milk containing: (:) no lutein, (B) 16 mg L1, and (-) 32 mg L1 of lutein, during ripening.
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Table 1 Chroma (C*) and hue angle (H*) values for Prato cheese during ripening time. Lutein concentration in milk (mg.L1)
C*
H*
Days
0 16 32
1
15
30
60
1
15
30
60
28.09 33.21 38.56
30.81 35.80 43.96
36.85 41.19 48.83
37.81 41.89 49.52
67.83 111.65 79.76
64.26 94.76 70.65
57.24 77.49 67.22
57.33 83.91 66.65
demonstrated similar values to the control treatment. The addition of lutein also changed the tonality of Prato cheeses; however, the H* values of control and cheese manufactured with a higher concentration of lutein were similar. The H* values indicate that control and higher lutein cheeses were orange, while cheese comprising low concentrations of lutein were yellow. Fig. 5 illustrates the difference between C* and H* in cheeses at day 60 of ripening. To confirm whether the difference in instrumental measurements of color due to the addition of lutein affected the acceptance of Prato cheeses, the global acceptance was evaluated by sensory analysis. Sensory evaluation was carried out by 40 non-trained individuals (23 women and 17 men between 20 and 43 years old) using a9-point scale on the last day of ripening (day 60). The average scores of global acceptance of Prato cheeses did not change (p 0.05) with replacement of annatto with lutein, and were 7.40, 6.95, and 7.35 for samples without lutein, and with 16 and32 mg L1 of lutein, respectively. These results corroborate the proximity in the color obtained by instrumental measurements, and indicate that lutein can be used as a natural pigment for Prato cheese. In addition, it is important to emphasize that even lutein presents higher cost than annatto; a small amount of this pigment (less than half concentration) was able to get desirable color of Prato cheese to consumers. Also, cheese containing lutein showed an additional advance related to its protection against AMD. 3.3. Ripening index, pH, and texture profile analysis Prato cheese is a ripened cheese often consumed after 30 days of ripening (Chaves & Gigante, 2016). Therefore, proteolysis is an important characteristic of this cheese. Ripening indices such as extension and depth of ripening were determined for cheeses with or without lutein since they involve the degradation of casein mainly by coagulant and low molecular weight nitrogenous
substances formed during ripening, respectively (Merheb-Dini, Garcia, Penna, Gomes, & da Silva, 2012). Ripening time influenced both indices (extension and depth of ripening) (p < 0.05) (Supplementary Material S3); however, there was no difference (p 0.05) in these parameters caused by lutein, indicating that this pigment did not change the ripening profile of Prato cheese. The pH is another important parameter of cheeses because it indicates changes in the ripening process caused by lactic acid bacteria. It depends on calcium phosphate and protein contents, salting conditions, and the salt sensitivity of the starter culture (Hou, Hannon, McSweeney, Beresford, & Guinee, 2012). For all treatments, the pH increased during ripening (p < 0.05), indicating proteolysis of the proteinous net of the cheese (Voigt et al., 2012). However, there was no difference (p 0.05) between the control or lutein-enriched cheese samples, and the average pH ranged from 5.2 to 5.5, as reported in other studies (Cichoscki, Valduga, Valduga, Tornadijo, & Fresno, 2002; Merheb-Dini et al., 2012). Therefore, replacement of annatto with lutein did not affect the pH increase during cheese ripening. To complete the characterization of Prato cheese, the texture profile of cheeses was evaluated during ripening. The texture profile is a very important parameter for characterizing cheeses as well as for consumer acceptance (Medeiros, Kongo, Borges, & Ponte, 2014). Firmness, springiness, and chewiness parameters decreased (p < 0.05) during ripening. These results can be explained by proteolysis of the casein network initially caused by the residual action of coagulants and then by proteolytic enzymes produced by lactic acid bacteria (LAB), which lead to a softer and lez et al., 2015). less elastic cheese (Ve The addition of lutein did not change (p 0.05) any texture profile parameters of the cheeses, thus the addition of lutein did not affect the ripening of Prato cheese (Table 3, in Supplementary Material S4).
4. Conclusion
Fig. 5. Graphical representation of C* and H* values for cheeses manufactured with milk containing: (A) 32 mg L1, (B) 16 mg L1 of lutein and (C) no lutein, at day 60 of ripening.
For the first time, the antioxidant activity of lutein carried by Prato cheese was evaluated during the 60 days of ripening. The cheese matrix formed by proteins and lipids protected the lutein during the ripening process, since there was no loss of lutein or its antioxidant capacity. Prato cheese with the highest concentration of lutein showed similar coloration (yellow-orange color) to traditional Prato cheeses produced with annatto, and the replacement of annatto with lutein did not influence the sensory acceptance of the cheese. The addition of lutein did not influence the maturation index, pH, and cheese texture profile compared to control during ripening. Prato cheese has significant potential as a lutein carrier, while maintaining the functional properties of this antioxidant. Furthermore, lutein was found to be innocuous to the cheese and may be used as a substitute for the annatto inducing functional properties of Prato cheeses.
D. Sobral et al. / LWT - Food Science and Technology 68 (2016) 349e355
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