Milk pre-treatment by high pressure homogenization in the manufacturing of “queso fresco” fortified with omega-3 fatty acids

Milk pre-treatment by high pressure homogenization in the manufacturing of “queso fresco” fortified with omega-3 fatty acids

LWT - Food Science and Technology 50 (2013) 629e633 Contents lists available at SciVerse ScienceDirect LWT - Food Science and Technology journal hom...

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LWT - Food Science and Technology 50 (2013) 629e633

Contents lists available at SciVerse ScienceDirect

LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt

Milk pre-treatment by high pressure homogenization in the manufacturing of “queso fresco” fortified with omega-3 fatty acids Sonia Calligaris a, *, Alessandro Gulotta a, Alexandra Ignat a, Daniela Bermúdez-Aguirre b, Gustavo V. Barbosa-Cánovas b, Maria Cristina Nicoli a a b

Dipartimento di Scienze degli Alimenti, Università degli Studi di Udine, via Sondrio 2/a, 33100 Udine, Italy Center for Nonthermal Processing of Food, Washington State University, Pullman, WA 99164-6120, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 March 2012 Received in revised form 16 July 2012 Accepted 24 July 2012

The aim of this study was to evaluate the feasibility of high pressure homogenization (HPH) as milk pretreatment in the process of manufacturing cheese fortified with omega-3 fatty acids. Raw milk was fortified with animal and vegetable sources of omega-3 fatty acids and homogenized at increasing pressure from 20 to 100 MPa. Treated milk was then used to prepare queso fresco, a soft and fresh Hispanic cheese. Cheese yield, moisture, fat content, and texture were evaluated after processing. The development of oxidation was also monitored during cheese storage at 4  C for up to 21 days. Pressure higher than 50 MPa was necessary to effectively incorporate omega-3 fatty acids in cheese and at the same time to reduce the quantity of oil lost in the whey. Homogenization treatments caused cheese quality attribute changes due to modifications induced by HPH process on native structures of milk. Cheeses obtained with homogenization showed higher moisture and yield and lower fat content than untreated cheeses. A decrease of texture parameters was also observed for homogenized samples. The source of omega-3 did not affect the cheese quality attributes. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Cheese Fortified food Omega-3 fatty acids High pressure homogenization

1. Introduction The fortification of food with essential polyunsaturated fatty acids (PUFA), such as omega-3 and omega-6 fatty acids, is an interesting and timely topic. It has been recognized that the daily intake of PUFA by populations in developed countries is below the recommended dose (Ferguson, Smith, & James, 2010; Iafelice et al., 2008). The health benefits associated with PUFA ingestion are several, such as reduction in cardiovascular diseases, antiinflammatory and anti-allergic effects, development and function of the brain, retina and nervous systems, protection against certain types of cancer (Iafelice et al., 2008; Kolanowski & Weibbrodt, 2008). In recent years the number of available PUFA fortified foods around the world has been rapidly increased in the attempt to address this nutritional deficiency of the population. Few studies have been reported on the fortification of milk and dairy products such as yogurt and cheese (Bermudez-Aguirre & Barbosa-Canovas, 2011; Kolanowski & Laufenberg, 2006; Ye, Cui, Taneja, Zhu, & Singh, 2009).

* Corresponding author. Tel.: þ39 0432558137; fax: þ39 0432558130. E-mail address: [email protected] (S. Calligaris). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2012.07.035

Naturally containing omega-3 fatty acids of animal (fish) or vegetable oils (i.e. flax-seed, canola, soybean) are generally chosen as ingredients for fortification. In the effort to produce fortified cheeses, the most cost-effective and easiest option is the direct addition of the nutrient to the milk before cheese-making. However, the oil separation at the milk surface makes its incorporation in the curd difficult and generally negligible (BermudezAguirre & Barbosa-Canovas, 2011). The effective incorporation of the nutrient in the curd, and thus in the cheese, can be obtained only by achieving a stable coexistence between the aqueous and the added oil phases. High pressure homogenization (HPH) is one of the most effective technologies applied to obtain stable emulsions. During the process, the fluid is forced to pass through a narrow gap in the homogenizer valve, where it is submitted to a rapid acceleration (Floury, Belletre, Legrand, & Desrumaux, 2004; Floury, Legrand, & Desrumaux, 2004). As a consequence, cavitation, shear stress, and turbulence are simultaneously inducted allowing the production of emulsions containing droplets from micro to nano scale (Freudig, Tesch, & Schubert, 2003; McClements & Rao, 2011). High pressure homogenization at levels around 20 MPa is currently applied by the dairy industry to prevent fat creaming during milk storage. The application of higher pressures has been

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proposed as milk pre-treatment before cheese-making for its ability to reduce microbial population in raw milk through mechanical disruption of cells (Escobar et al., 2011; Lanciotti et al., 2004; Vannini et al., 2008). Based on literature, cheeses obtained from homogenized milk are expected to have physicochemical and sensory properties different from cheeses processed with conventional methods (Escobar et al., 2011; Lodaite, Chevalier, Arnaforte, & Kelly, 2009; Vannini et al., 2008; Zamora, Ferragut, Jaramiillo, Guamis, & Trujillo, 2007; Zamora, Ferragut, Juan, Guamis, & Trujillo, 2011). In particular, changes in cheese yield, composition (moisture, fat and protein content) and texture have been reported when milk was treated by HPH. These changes have been attributed to modification of the functional properties of milk proteins as well as the disruption of milk fat globules as a consequence of the passage through the homogenization valve (Kelly, Huppertz, & Sheehan, 2008). The effects of HPH on cheese quality attributes are affected by the homogenization pressure and number of passes through the homogenization valve: as more intense the treatment is, more differences in the physicochemical and structural properties of the cheese have been observed (Escobar et al., 2011; Zamora et al., 2007). The aim of the present research was to study the feasibility of HPH as milk pre-treatment during cheese-making of fortified queso fresco with omega-3 fatty acids. This cheese is a fresh and soft Hispanic-style cheese popular in Latin-America and US markets. Before cheese-making, milk was fortified with animal or vegetable sources (cod liver oil or flax-seed oil) and treated at increasing pressure from 20 to 100 MPa. The effects of HPH on cheese yield, moisture and fat content as well as on texture were evaluated. Since the main issue in producing omega-3 fatty acids fortified products is related to the oxidative stability of the incorporated PUFA, the oxidation degree was also monitored by measuring the changes of peroxide value during storage of cheese at 4  C.

milk was added with 0.05 g/100 g of sodium azide (Carlo Erba, Milano, Italy). Control cheeses were prepared following the same procedure but using i) milk added with fish or flax-seed oil that was not homogenized; ii) milk that was homogenized at increasing pressure but not added with fish or flax-seed oil. The samples were packed in sterile plastic bags and stored at 4  C for up to 21 days. 2.2. Moisture content Moisture content was determined by gravimetric method according to AOAC (AOAC, 2000). 2.3. Fat content The fat content was determined following the Schmidte BonzynskyeRatzalaff method (FIL-IDF Standard 5A method, 1969). Aliquots of 1.5 g of cheese were decomposed with 10 mL of concentrated hydrochloric acid (37 mL/100 mL) (Carlo Erba, Milano, Italy) for 40 min in a water bath at 90e95  C. After cooling the digested samples at room temperature using a water bath, 10 mL of ethanol 96 mL/100 mL (Carlo Erba, Milano, Italy) were added and the lipids were extracted with 50 mL of diethyl ether/ pentane mixture (1:1 mL/mL) (Carlo Erba, Milano, Italy). The alcoholewater phase was removed, and the etheric phase was washed 3 times using 50 mL ethyl ether/petroleum ether mixture (1:1 mL/mL). Subsequently the etheric phase was recovered and the solvent was evaporated using a rotary evaporator (Buchi, mod 110, Flawil, Switzerland). After drying in oven at 70  C, the fat content was determined gravimetrically. In case of whey, 10 g of sample was weighted and added with 1.5 mL of ammonia 28 mL/100 mL (Carlo Erba, Milano, Italy) and 50 mL of ethyl ether/pentane mixture (1:1 mL/mL). Afterward, the alcoholewater phase was treated as described above for the determination of cheese fat content.

2. Material and methods 2.4. Cheese yield 2.1. Milk processing and cheese-making Raw whole bovine milk was obtained from a local dairy farm (Latteria Sociale di Coderno, Udine, Italy). The milk was stored at 4  C until used (maximum 6 h of storage). Aliquots of 2 kg of refrigerated milk was heated to 20  C and added with 20 g of cod liver oil (named fish oil in the text) (Marco Viti Farmaceutici SpA, Vicenza, Italy) or flax-seed oil (Solimè, Cavriago, Italy). According to producer indications, total omega-3 fatty acids in cod liver oil were 27.5 g/100 g and in flax-seed oil were 53.4 g/100 g. The oil final concentration in milk was 1 g/100 g. The oil was initially dispersed by using a high speed homogenizer (Polytron, PT 3000, Cinematica, Littau, Switzerland) for 2 min at 3600 rpm. The samples were then immediately treated by using a lab-scale high pressure homogenizer NS1001L-PANDA 2K (GEA Niro Soavi S.p.A., Parma, Italy) at increasing pressure from 20 to 100 MPa. The lab-scale high pressure unit NS1001L-PANDA 2K is a two stage homogenizer whose valves are composed of a ceramic ball (GEA Niro Soavi S.p.A., Parma, Italy). The first valve is the actual homogenization stage and was set at increasing pressure from 20 to 100 MPa. The second valve was set at the constant value of 5 MPa. The sample was homogenized at flow rate of 10 L/h. The inlet temperature of milk was 20  C; the outlet temperature never exceeded 40  C. The fortified and homogenized milk was subdivided in 500 g aliquots and used to prepare queso fresco following the methodology proposed by Bermudez-Aguirre and Barbosa-Canovas (2011). To the only aim to avoid microbial spoilage during the experiments,

Cheese yield was calculated as the percentage ratio between the mass of the cheese (g) over mass of initial milk (g). 2.5. Texture analysis Texture profile analysis (TPA) tests were performed by using an Instron Universal Machine model 4301 (Instron International Ltd, High Wycombe, UK) with a 1 kg load cell. Cheese was cut into 2  2  2 cm cubes. Samples were equilibrated at room temperature for 30 min before analysis. Cheese cubes were compressed at 50% of their original height using a 5-cm-diameter cylindrical probe moving at a displacement speed of 5 cm/min. A 5 s pause was allowed between the first and the second compression. TPA parameters were determined: hardness (maximum force during first compression (N)), cohesiveness (ratio between positive areas of a TPA plot: dimensionless) and gumminess (product of hardness and cohesiveness: N). 2.6. Peroxide value Lipids were extracted from the cheese following the methodology described by Kristensen, Orlein, Mortensen, Brockhoff, and Skibsten (2000). About 10 g of each cheese was transferred to a 100 mL plastic centrifuge tube and 50 mL of methanole isooctaneeethyl ether solution (1:2:2 mL/mL) (Carlo Erba, Milano, Italy) was added. The sample was homogenized using a high speed homogenizer (Polytron, PT 3000, Cinematica, Littau, Switzerland),

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added with 5 mL of CaCl2 (1 mmol/L) and centrifuged at 924  g for 15 min (Beckman, Avanti Centrifuge J-25, Palo Alto, CA, USA). The supernatant was transferred in a 200 mL distilling flask and the solvent was separated under vacuum using a rotary evaporator (Buchi, model 110, Flawil, Switzerland). Peroxide value was determined following the method of Shanta and Decker (1994). Lipid samples (0.02 g) were weighted into 20 mL volumetric flask and then 9.8 mL of di-chloromethane/ methanol (Carlo Erba, Milano, Italy) solution (7:3 mL/mL) was added and vortexed for 5 s. For each sample, 0.05 mL of ferrous ion solution (prepared through the mixture of BaCl2 0.132 mol/L and FeSO4 0.144 mol/L) and 0.05 mL of 3.95 mol/L ammonium thiocyanate solution were added to the flask and vortexed for 5 s. After 5 min of incubation at room temperature, absorbance was measured at 510 nm with a spectrophotometer (Shimadzu, UV2501PC, Japan). The peroxide value (PV) expressed as millimoles of oxygen per kilogram of fat was calculated by using the follow equation:

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Table 2 Moisture content (g/100 g) of cheeses made from fortified or not-fortified milk by the addition of 1 g/100 g of fish or flax-seed oil and homogenized at pressure from 0 to 100 MPa. Homogenization pressure (MPa)

Moisture (g/100 g)

0 20 50 100

64.8 64.1 72.1 70.4

Unfortified milk    

1.3a 1.2a 2.1b 1.5b

Milk fortified with fish oil 65.4 64.2 69.4 68.5

   

1.3a 1.5a 0.9b 0.8b

Milk fortified with flax-seed oil 63.92 65.10 66.55 66.73

   

0.9a 1.1ab 0.8b 0.5b

Mean  S.D. (n ¼ 3). a,b Values followed by same letter in the same column are not significantly different (p > 0.05).

Table 1 shows the cheese yield of the samples obtained from milk previously homogenized at increasing pressure from 0 to 100 MPa. The yield values of both unfortified and fortified samples increased as the homogenization pressure also increased, specially in the range of 20e50 MPa. No further changes of the cheese yield were observed at 100 MPa. These data are consistent with those reported by Lanciotti et al. (2004), Vannini et al. (2008), Escobar et al. (2011). The cheese yield increase as a consequence of HPH has been associated to the increase of moisture content in the product. It has been suggested that HPH induces the partial denaturation of whey proteins increasing their capacity to bind water

(Dumay, Kalichevsky, & Cheftel, 1994; Heremans & Smeller, 1998; Lanciotti et al., 2004; Vannini et al., 2008; Venir, Marchesini, Biasutti, & Innocente, 2010). Moisture content of the cheeses are shown in Table 2. As expected, fortified samples prepared from unprocessed milk (pressure ¼ 0 MPa) were not significantly different from the corresponding unfortified cheeses. On the contrary, homogenization pressures higher than 20 MPa were responsible for a significant increase in moisture content. Such moisture gain was smaller in the case of the fortified samples than in the corresponding unfortified ones. In all cases, no significant differences (p > 0.05) were observed among 50 and 100 MPa treated samples. These data suggest that the changes in yield observed for the fortified cheeses could be reasonably ascribed to the increase of moisture together with the concomitant fat incorporation. The latter is evidenced by the fact that, considering samples obtained from milk treated at the same homogenization pressure, the calculated non-fat dry matters of fortified and unfortified products are comparable (data not shown). Table 3 shows the fat content on dry basis of the samples under study. HPH induced a significant decrease of the fat content in unfortified products. This effect was well evident already at 20 MPa. As it is well known, HPH disrupts milk fat globules and modifies their surface layer, reducing the fat entrapment in the curd (Escobar et al., 2011; Kelly et al., 2008). Increasing the operative pressure, the fat content of the unfortified products slightly increased probably due to the combined effect of homogenization on fat globule and milk proteins. It can be hypothesized that the HPH-induced protein unfolding enhanced their emulsifying capacity allowing fat to be more entrapped in the curd (Zamora et al., 2007). Moving to fortified cheeses, the fat content of the samples obtained from unprocessed milk was not significantly different from that of the corresponding unfortified ones. This confirms that the simple addition of the oil to the milk did not allow its incorporation in the curd.

Table 1 Yield of cheeses made from fortified and not-fortified milk by the addition of 1 g/100 g of fish or flax-seed oil. These samples were homogenized at pressure from 0 to 100 MPa.

Table 3 Fat content (g/100 g dry basis) of cheeses made from fortified and not-fortified milk by the addition of 1 g/100 g of fish or flax-seed oil and homogenized at pressure from 0 to 100 MPa.

PV ¼ ½ðAs  Ab Þ$m=ð55:84$m0 $2Þ where As is the absorbance of the sample, Ab is the absorbance of the blank, m is the slope of the calibration curve of Fe3þ concentration vs absorbance, m0 is the mass in grams of the sample and 55.84 is the atomic weight of iron. 2.7. Data analysis Each experiment was performed at least in duplicate and measurements performed at least in triplicate. Data are reported as mean value and relevant standard deviation (S.D.). Analysis of variance (ANOVA) was performed by using Statistica for Windows (ver. 5.1, Statsoft Inc., Tulsa, USA, 1997). A Tukey’s Test was used to evaluate significance of differences among means (p < 0.05). 3. Results and discussion

Homogenization pressure (MPa)

Yield (g/100 g)

0 20 50 100

21.9 23.8 28.7 28.3

Unfortified milk    

1.1a 1.3a 1.5b 1.7b

Milk fortified with fish oil 22.1 24.6 27.8 27.1

   

0.9a 1.0a 1.5b 1.2b

Milk fortified with flax-seed oil 22.3 24.1 27.3 27.5

   

1.4a 1.0a 1.3b 1.1b

Mean  standard deviation (S.D.) (n ¼ 3). a,b Values followed by same letter in the same column are not significantly different (p > 0.05).

Homogenization pressure (MPa)

Fat content (g/100 g dry basis)

0 20 50 100

50.8 40.3 43.8 44.9

Unfortified milk    

1.3a 1.1b 0.9c 1.4c

Milk fortified with fish oil 52.5 43.0 59.7 49.4

   

0.9a 0.5b 2.0c 1.5a

Milk fortified with flax-seed oil 51.2 42.9 53.3 48.7

   

1.1ac 0.8b 1.5a 0.8c

Mean  S.D. (n ¼ 3). a,b,c,d Values followed by same letter in the same column are not significantly different (p > 0.05).

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10

Table 4 Fat content (g/100 g dry basis) of the whey separated after cheese-making of cheeses made from fortified and not-fortified milk by the addition of 1 g/100 g of fish or flaxseed oil and homogenized at pressure from 0 to 100 MPa. Fat (g/100 g dry basis)

0 20 50 100

4.0 7.3 6.5 6.8

Unfortified milk    

Milk fortified with fish oil

0.2a 0.4b 0.6b 0.5b

4.7 8.5 4.4 5.1

   

8

Milk fortified with flax-seed oil

0.5a 0.4b 1.1a 0.7a

4.4 10.2 4.9 5.4

   

Peroxide value (meqO2 /kg fat)

Homogenization Pressure (MPa)

0.9a 1.5b 1.3a 0.8a

Mean  S.D. (n ¼ 3). a,b Values followed by same letter in the same column are not significantly different (p > 0.05).

By applying HPH at 20 MPa the fat content on dry basis slightly increased as compared to the correspondent unfortified samples, highlighting that certain oil incorporation in the curd occurred. At 50 MPa, such incorporation resulted significantly higher. However, further pressure increase (100 MPa) caused a reduction of fat retention probably due to advanced modifications of milk native structures leading to a curd network able to entrap less quantity of fat. Cheese fat content data are consistent with those of whey (Table 4). The homogenization treatment of unfortified milk induced the increase of fat in the whey. Referring to fortified products, it is evident that part of the oil added to milk processed at 20 MPa remained in the whey after cheese-making. On the contrary, samples subjected to higher homogenization conditions showed a reduced oil loss in the whey. These results highlighted the efficiency of homogenization at 50 MPa to stabilize oil droplets in the milk before cheese-making. It should be noted that slight differences among samples fortified with fish oil or flax-seed oil were observed indicating that HPH efficacy was not affected by the oil source. The texture characteristics of the products were finally determined by using TPA analysis. Table 5 shows values of hardness, cohesiveness and gumminess of the cheeses obtained from unfortified milk and milk fortified with fish oil and homogenized at increasing pressure. Not significantly different results (p > 0.05) in the texture characteristics, expressed by means of the above mentioned parameters, were observed. It should be noted that cheeses fortified with flax-seed oil resulted also not significantly different from the respective sample reported in Table 5 (data not shown). Thus fortification did not affect cheese texture characteristics. It can be hypothesized that the differences observed in terms of fat content among fortified and unfortified cheesed were not enough to induce texture modifications. Observing Table 5, it is evident that the homogenization was responsible for a decrease of all the considered texture parameters. These results are in agreement with Escobar et al. (2011) and can be linked to the changes in cheese moisture content as well as protein

6

4

2

0 0

5

10

15

20

25

30

Storage time (days) Fig. 1. Changes of the peroxide value of chesses made from fortified milk by the addition of 1 g/100 g of fish (A) or flax-seed ( ) oil and homogenized at 50 MPa. Each experiment was carried out by duplicate. Bars represent the standard deviation (n ¼ 3).

structure as a consequence of the passage through the homogenization valve. The stability of fortified cheeses against oxidation during storage at 4  C was finally evaluated. To this purpose, 50 MPa fortified cheeses were considered. No significant differences in peroxide values (p > 0.05) were detected between unprocessed and homogenized samples indicating that homogenization was not responsible for promoting the development of oxidation (data not shown). However, observing Fig. 1, samples containing fish oil showed a sharp increase in the peroxide value as compared to those fortified with flax-seed oil. Interestingly, flaxseed fortified products resulted quite stable against oxidation, highlighting that the development of rancid off-flavor are expected to be very low during storage at 4  C for up to 28 days. This result can be reasonably attributed to the different degree of unsaturation and the presence of antioxidants, mainly vitamin E and vitamin A, of flax-seed oil (Barrett, Porter, Marando, & Chinachot, 2011). These results confirmed that the choice of omega-3 fatty acid source has a great impact in determining product stability. In the case of fish oil fortified cheese, the improvement of stability could be obtained by designing proper packaging conditions and/or by adding antioxidant molecules in the formulation.

Table 5 Hardness, cohesiveness and gumminess of cheeses made from milk unfortified and fortified with fish oil and homogenized at pressure from 0 to 100 MPa. Homogenization pressure (MPa) 0 20 50 100

Hardness (N) Unfortified 7.53 4.35 3.25 4.61

   

0.44a 0.75b 0.63c 0.42bc

Cohesiveness Fortified 7.03 4.81 3.16 4.21

   

0.45a 0.80b 0.70c 0.41bc

Unfortified 0.55 0.36 0.41 0.42

   

0.04a 0.06b 0.03b 0.06b

Mean  S.D. (n ¼ 3). a,b,c Values followed by same letter in the same column are not significantly different (p > 0.05).

Gumminess (N) Fortified 0.58 0.34 0.43 0.39

   

0.05a 0.07b 0.03b 0.06b

Unfortified 4.10 1.82 1.52 1.91

   

0.50a 0.25b 0.65b 0.82b

Fortified 4.06 1.74 1.46 1.90

   

0.50a 0.23b 0.65b 0.84b

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4. Conclusions Results reported in this study highlighted that queso fresco obtained from milk previously subjected to HPH treatment showed different characteristics as compared to those obtained by unprocessed milk. HPH treated cheeses mainly differed for moisture, fat content and textural properties. However, when fish oil or flax-seed oil were added to the milk, the HPH treatment resulted a reliable technological solution in the attempt to produce queso fresco fortified with omega-3 fatty acids. Homogenization at 50 MPa pressure was the process that allowed the highest fat retention and these results are not affected by the source of omega-3 fatty acids. Since slight differences of product characteristics were observed among 50 and 100 MPa treated samples, additional considerations should be taken in defining the best processing conditions to be applied to produce fortified queso fresco. For instance, the effect of HPH on milk microbial population could be a point of decision. Even though this study has been focused on queso fresco fortification, the proposed approach could offer an interesting way to effectively incorporate oils rich in omega-3 fatty acids into other kind of cheeses. The advantage could be that the use of HPH as milk pre-treatment could be easily introduced in the conventional cheese-making. However, the changes induced by HPH on physicochemical and sensory attributes of cheeses should be carefully considered.

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