Prato cheese as suitable carrier for Lactobacillus acidophilus La5 and Bifidobacterium Bb12

International Dairy Journal 52 (2016) 10e18

Contents lists available at ScienceDirect

International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj

Prato cheese as suitable carrier for Lactobacillus acidophilus La5 and Bifidobacterium Bb12 K.S. Chaves, M.L. Gigante* Department of Food Technology, Faculty of Food Engineering, University of Campinas, UNICAMP, 13083-862, Campinas, SP, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 September 2014 Received in revised form 20 August 2015 Accepted 22 August 2015 Available online 6 September 2015

To assess Prato cheese as suitable carrier for probiotic bacteria, four cheeses were produced. Control cheese contained only starter culture, whereas the others contained starter culture and the probiotic cultures Lactobacillus acidophilus La5 and Bifidobacterium Bb12 either separately or in combination. Bacterial viability, physicochemical composition, proteolysis, and texture profile were assessed over 60 days of storage. The addition of microorganisms together or separately did not affect the characteristics of Prato cheese. On storage, the cheeses showed increased proteolysis, lower firmness, and the probiotic cheeses presented counts higher than 106 cfu g
1. The viability of probiotics during in vitro gastrointestinal simulation, including the effect of the cheese matrix, was also assessed. The probiotic bacteria showed resistance to loss of viability during in vitro gastrointestinal simulation. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The demand for probiotic functional foods is growing rapidly due to increased awareness of consumers about the impact of food on health (Tripathi & Giri, 2014). This high demand has motivated a large number of studies focusing on the incorporation of probiotics into dairy and non-dairy products (Albenzio et al., 2013), such as fermented dairy products (Donkor, Nilmini, Stolic, Vasiljevic, & Shah, 2007; Oliveira, Sodini, Remeuf, & Corrieu, 2001), ice cream (Akin, Akin, & Kirmaci, 2007), chocolates (Possemiers, Marzorati, Verstraete, & Van de Wiele, 2010), vegetables (Lavermicocca et al., 2005), and beverages (Shimakava, Matsubara, Yuki, Ikeda, & Ishikawa, 2003). The choice of a food system to deliver adequate doses of probiotics at the time of consumption is a challenging task. Several factors during processing and storage affect the viability of probiotics, which, in turn, may have a negative impact on the quality and sensory properties of the products. Some authors have suggested that cheese has certain advantages as a carrier of probiotics when compared with other fermented milk products due to its physicochemical characteristics. Several factors, including high pH, low acidity, high buffering capacity, and an anaerobic environment created by the protein-fat matrix of cheese contributes to

* Corresponding author. Tel.: þ55 19 35213993. E-mail address: [email protected] (M.L. Gigante). http://dx.doi.org/10.1016/j.idairyj.2015.08.009 0958-6946/© 2015 Elsevier Ltd. All rights reserved.

the maintenance of probiotics viability throughout the product shelf-life and during passage through the gastrointestinal tract (Bergamini, Hynes, Palma, Sabbag, & Zalazar, 2009; Ross, Fitzgerald, Collins, & Stanton, 2002; Stanton et al., 1998; Vinderola, Prosello, Ghiberto, & Reinheimer, 2000). It is widely recognised that the development of typical characteristics in cheeses is related to its microbiota during ripening. Thus, the addition of probiotic microorganisms influences the biochemical events during ripening, such as proteolysis and lipolysis, for producing enzymes that can affect the development of cheese flavour, aroma and texture (Bergamini et al., 2009; Karimi, Mortazavian, & Da Cruz, 2011; Ong, Henriksson, & Shah, 2006; Vinderola & Reinheimer, 2000). The incorporation of lactobacilli and bifidobacteria in different cheeses has been widely studied,
langer, & Vuillemard, 1999; Mc such as Cheddar (Daigle, Roy, Be Brearty et al., 2001; Milesi, McSweeney, & Hynes, 2008; Ong & Shah, 2009; Ong et al., 2006; Scheller & O'Sullivan, 2011), Kasar €
s Argentino (Bergamini, (Ozer, Uzun, & Kirmaci, 2008); Pategra Hynes, & Zalazar, 2006), Canestro Pugliese (Corbo, Albenzio, De Angelis, Sevi, & Gobbetti, 2001), Cottage (Abadía-García et al., 2013), Minas Frescal (Buriti, Rocha, & Saad, 2005), Gouda (Gomes, Vieira, & Malcata, 1998), and Scamorza (Albenzio et al., 2013). In general, these studies have focused on the maintenance of probiotic viability during storage and at the end of the product shelf-life. Few studies have reported, even in vitro, the survival of probiotics during passage through the gastrointestinal (GI) tract when delivered within the cheese matrix.

K.S. Chaves, M.L. Gigante / International Dairy Journal 52 (2016) 10e18

GI tract barriers, including gastric juice, hydrolytic enzymes, and bile salts are the main challenges to the survival of probiotics (Liu et al., 2007). Tolerance of probiotics, both in vitro and in vivo, during the passage through the GI tract is strongly influenced by intrinsic characteristics of each strain and by the physicochemical characteristics of food (Blaiotta et al., 2013; HernandezeHernandez et al., 2012). Although the evaluation of probiotics survival during in vitro gastrointestinal simulation cannot predict responses on the functionality of the microorganisms in human body, it demonstrates the protective effect of the food matrix on probiotics survival during the passage through the GI tract (Schillinger, Guigas, & Holzapfel, 2005). A review published recently pointed out a
, Daniel, & Pot, 2013). Appromising future for probiotics (Foligne plications will be both in the food domain, affecting large parts of the population, as well as very specific disease situations, in which probiotics can play a true therapeutic role for a limited number of patients, especially probiotic products composed of multiples species. In the field of dairy products, fermented milk and fresh cheeses are the most marketed products, which are generally produced with only one bacterial species. The aim of this study was to evaluate the effect of Lactobacillus acidophilus La5 and Bifidobacterium Bb12, separately or in combination, during ripening on the viability of microorganisms, and physicochemical characteristics of Prato cheese, a semi-hard and medium moisture cheese. The cheese matrix effect on the viability of the probiotics during simulation of the passage through GI tract was also assessed. 2. Materials and methods 2.1. Preparation of culture Lyophilised probiotic cultures (entire package, 25 g freeze dried culture) of Lb. acidophilus La5 and Bifidobacterium Bb12 (Chr. Hansen, Valinhos, SP, Brazil) were rehydrated in 1 L of sterile reconstituted skim milk (10%, w/v) at 7  C for 1 h. The rehydrated probiotic culture (0.5 or 1%, v/v) was added to 50 L of milk at 35  C. Milk was allowed to stand for 40 min and probiotic viability was assessed prior to the addition of the coagulant. Lyophilised culture of Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris (R 704; Chr. Hansen) was reactivated in 10% (w/v) sterile reconstituted skim milk, incubated at 30  C for 12 h, and used in cheese manufacture. 2.2. Cheese manufacture Cheese was produced by a traditional manufacturing method as described by Mazal, Vianna, Santos, and Gigante (2007), and the experiment was carried out in triplicate. For each process, 200 L of heat-treated milk (68  C for 2 min) were cooled to 35  C and divided into four equal parts to be used for Prato cheese manufacture. The following protocols were carried out when manufacturing the Prato cheese: (i) a control using 1% (v/v) starter culture (Lc. lactis ssp. lactis and Lc. lactis ssp. cremoris); (ii) 1% (v/v) starter culture and 1% (v/v) Lb. acidophilus La5; (iii) 1% (v/v) starter culture and 1% (v/v) Bifidobacterium Bb12; (iv) 1% (v/v) starter culture and 0.5% (v/v) each of Lb. acidophilus La5 and Bifidobacterium Bb12. Milk was allowed to stand for 40 min and then 50% calcium chloride solution (250 mg kg
1), annatto dye (80 mg kg
1), and coagulant (HA-LA 1175 100% chymosin; Chr. Hansen) sufficient to coagulate the milk within 35 min were added. The curd was cut into 1.0 cm cubes and submitted to slow continuous mixing for 15 min, followed by heating the curd to 42  C by increasing 1  C every 3 min. All whey was drained off and the curd was placed into rectangular plastic moulds (0.5 kg) and pressed at room temperature (0.1 MPa for 15 min; 0.1 MPa for 15 min; 0.24 MPa for 30 min;

11

and 0.31 MPa for 90 min). The cheeses were further fermented for 2.5 h at room temperature and salted in brine (20%) for 10 h at 5  C. The cheeses were then dried at 12  C for 48 h and vacuum-packed into heat-shrinkable plastic bags and stored at 12  C for 60 days. 2.3. Physicochemical composition Heat-treated milk was characterised for pH (pH meter DM22Digimed, S~ ao Paulo, SP, Brazil), total solids (AOAC., 2006a), fat by the Gerber method (AOAC., 2006b), ash content (AOAC., 2006c) and total nitrogen (TN; AOAC, 2006e). Total protein content was calculated by multiplying TN by 6.38. The activities of the enzymes alkaline phosphatase (AOAC, 2006g) and lactoperoxidase (LANARA, 1981) were determined to check the efficiency of heat treatment. Cheese samples were characterised 1 day after manufacture (24 h after packaging) for pH measurements, titratable acidity (AOAC, 2006h), moisture content (AOAC, 2006f), and TN content (AOAC, 2006d). Total protein content was calculated as previously described. Fat content was determined by the Gerber method (BSI., 1989) and salt by the Volhard method (Richardson, 1985). 2.4. Cheese characterisation during ripening After 1, 7, 14, 28, 40, and 60 days of refrigerated storage, the cheeses were evaluated for pH, titratable acidity, moisture, proteolysis, firmness, concentration of organic acids and viability of microorganisms. The pH was determined by introducing the electrode directly into the sample, and moisture was determined by oven drying according to AOAC (2006f). Proteolysis was assessed by soluble nitrogen (SN) at pH 4.6, expressed as percentage of TN [pH 4.6-SN (% TN)] and soluble nitrogen in 12% trichloroacetic acid (12% TCA-SN) expressed as percentage of TN [12% TCA-SN (% TN)], determined by the macro-Kjeldahl method according to Bynum and Barbano (1985). The electrophoretic profile of the casein fractions was determined according to Otte, Zakora, Kristiansen, and Qvist (1997) and Ortega, Albillos, and Busto (2003) with modifications, using a Beckman Coulter P/ACE MDQ capillary electrophoresis system and 32 Karat software (BeckmanCoulter, Santana de Parnaíba, SP, Brazil). The separation was carried out using a fused silica capillary with 57 cm  75 mm internal diameter (50 cm effective length). A sample of 20 mg of cheese was dissolved in 1 mL of a solution containing 10 mM sodium phosphate, 8 M urea, and 10 mM dithiothreitol at pH 8.0, and allowed to stand for about 1 h. The sample was then filtered (0.45 mm) and injected for 5 s at injection rate of 3.45  10
3 Pa. The separation was carried out at 18.5 kV and 23  C and detection was done at 214 nm for 60 min. Between runs, the capillary was conditioned by washing for 5 min with NaOH (0.5 M), pure water, and running buffer (10 mM sodium phosphate, 6 M urea, and 0.05% hydroxypropyl methylcellulose, pH 3.0). Firmness was evaluated using a TA-XT2 texture analyser (Stable Micro Systems Ltd. Godalmning, Surrey, UK) with 35 mm diameter aluminium probe. Sampling was carried out according to Mazal et al. (2007), and the experiments were carried out in octuplicate. For testing, the cheese samples were cut into cylinders of 2 cm diameter and 2.4 cm height, placed in waterproof bags and kept in a water bath at 10  C for at least 4 h for temperature stabilisation. The test speed was 100 mm per min, and samples were compressed to 40% of their original height, repeated within 5 s. Production of lactic acid and acetic acid was determined using high performance liquid chromatography (HPLC) as described by Marsili, Ostapenko, Simmons, and Green (1981) with modifications. Sample (1 g) was dissolved in 50 mL 4.5 mM sulphuric acid containing 15 mg kg
1 of boric acid as an internal standard. The sample was homogenised using an Ultra Turrax for 1 min, and then kept

12

K.S. Chaves, M.L. Gigante / International Dairy Journal 52 (2016) 10e18


nezunder slow stirring conditions for 15 min (Izco, Tormo, & Jime Flores, 2002). After stirring, the sample was centrifuged at 18,000 g for 4 min at 4  C and the supernatant was filtered through PVDF 0.45 mm membrane (Millipore Corporation, Bedford, MA, USA). Elution was in an isocratic mode at a flow rate of 0.5 mL min
1 with 4 mM H2SO4 as mobile phase. Injection volume was fixed at 20 mL for all trials. Analyses were carried out on a Shimadzu LC-10 HPLC system (Columbia, MD, USA), consisting of 10 LC-VP pump, CTO-10AS VP column oven, with SPD-M20A diode array detector, SCL-10A VP interface, and Class VP software. Aminex HPX-87H column (300  7.8 mm) (BioRad, Hercules, CA, USA) maintained at 35  C was used for the analysis. The diode array detector was operated between 200 and 800 nm. The chromatograms were recorded at 210 nm for quantitative analysis. 2.5. Bacterial viability Preliminary studies were carried out to define a selective methodology for enumeration of Lc. lactis sp., Lb. acidophilus, Bifidobacterium and non-starter lactic acid bacteria (NSLAB) from cheeses (data not shown). Cheese samples (25 g) were transferred to stomacher bags (Stomacher® 400 Classic Standard Bag; Seward Limited, Worthing, UK) with 225 mL sodium citrate (2%) for homogenisation (10 min), and subjected to serial dilutions in 0.1% peptone water (w/v). Lc. lactis was enumerated on M17-lactose agar incubated at 30  C for 48 h under aerobic conditions (Terzaghi & Sandine, 1975). Lb. acidophilus was enumerated on MRS sorbitol agar, with glucose replaced by sorbitol (10 g L
1) incubated at 45  C for 72 h under anaerobic conditions (Oberg, Moyes, Domek, Brothersen, & McMahon, 2011). Bifidobacterium was enumerated on MRS-NNPL agar (neomycin sulphate, nalidixic acid, paromomycin sulphate, and lithium chloride) incubated at 37  C for 72 h under anaerobic conditions (Ong et al., 2006). NSLAB were plated onto reinforced clostridium agar (RCA) medium with bromocresol green and vancomycin, and incubated under anaerobic conditions at 37  C for 48 h. RCA agar was used as a base medium, with pH adjusted to 5.5 with HCl (1 M) prior to sterilisation (121  C for 15 min). An aliquot of 20 mL bromocresol green sterilised stock solution (0.2%, w/v) was added into 1 L base medium prior to use. Vancomycin stock solution (2%, w/v) was sterilised by filtration through 0.22 mm membrane (Millipore Corporation) and 0.5 mL solution was added into 1 L base medium at the time of analysis (Darukaradhya, Phillips, & Kailasapathy, 2006). Probiotic and starter culture cannot grow in the RCA medium with bromocresol green and vancomycin. 2.6. Simulation of gastrointestinal conditions Viability of Lb. acidophilus La5 and Bifidobacterium Bb12 during gastrointestinal simulation was assessed after 28 and 60 days of Prato cheese storage using the culture media and incubation conditions previously described. The gastric and intestinal juices were prepared according to Mozzi, Gerbino, Font de Valdez, and Torino (2009) and Picot and Lacroix (2004), with modifications. The artificial gastric juice (AGJ) consisted of potassium chloride (1.12 g L
1), sodium chloride (2.0 g L
1), calcium chloride (0.11 g L
1), and monobasic potassium phosphate (0.4 g L
1), which was sterilised (121  C for 15 min). Immediately before use, mucin (3.5 g L
1) and pepsin (0.26 g L
1) were added, and the pH was adjusted to 2.0 with 1 M HCl. The artificial intestinal juice (AIJ) was prepared by adding pancreatin to the AGJ solution to reach a final concentration of 1.95 g L
1. Then, pH was adjusted to 7.0 with 1 M NaHCO3. Cheeses were ground in industrial grinder (model TA2BIVMF60N5; BIMG Brasil Indústria de M
aquinas, Metvisa) and samples (10 g) were placed in Erlenmeyer flasks containing 100 mL of AGJ (pH 2.0) and

incubated at 37  C in a metabolic bath with reciprocal agitation of 120 rpm. The viability of probiotic microorganisms was assessed after 0, 60, and 120 min of exposure to AGJ. After 120 min, pancreatin solution was added and the pH was adjusted to 7.0 with 1 M NaHCO3 for simulation of the intestinal conditions. Probiotic viability was assessed after 300 min of contact with AIJ. Bile tolerance procedure was carried out according to Gerez, Font de Valdez, Gigante, and Grosso (2012) at 0.5% bile solution (pH 7.0). Cheese were crushed and samples (10 g) were placed in Erlenmeyer flasks containing 100 mL of bile solution, and incubated at 37  C for 300 min in a metabolic bath with reciprocal agitation of 120 rpm. Probiotic viability was assessed after 0, 60, and 300 min of exposure to the bile solution. 2.7. Experimental design and statistical analysis The experiments were carried out in a complete randomised block design with a 4  6 factorial arrangement and three replications. The effect of type of cheese (four levels of variation: control Prato cheese, Prato cheese with Lb. acidophilus La5, Prato cheese with Bifidobacterium Bb12, and Prato cheese with Lb. acidophilus La5 and Bifidobacterium Bb12); storage time (six levels of variation: 1, 7, 14, 28, 40, and 60 days after manufacture), as well as the interaction of these factors, on the physicochemical characteristics of the cheeses was assessed by analysis of variance (ANOVA) and Tukey's test for comparison between means at the 5% significance level. The probiotic viability during in vitro GI simulation and tolerance to bile salts (0.5%) for each probiotic microorganism tested was subjected to ANOVA at the 5% significance level, considering the independent effects of the type of probiotic cheese and the time of exposure to in vitro GI simulation or bile, after 28 and 60 days of storage. Data were analysed using the software STATISTICA 7.0 (StatSoft Inc, Tulsa, OK, USA). 3. Results and discussion 3.1. Characterisation of raw material The milk used for cheese production met the physicochemical requirements established by Brazilian legislation (Brasil, 2011), with 12.15 ± 0.33% total solids, 3.5 ± 0.40% fat, 3.31 ± 0.09% protein, 0.68 ± 0.06% ash, and pH 6.80 ± 0.02. The heat treatment applied to milk was adequate, showing a negative alkaline phosphatase activity and positive lactoperoxidase activity. 3.2. Viability of probiotic microorganisms and physicochemical composition of the cheeses As shown in Table 1, the manufacturing process was suitable for obtaining probiotic Prato cheeses once bacteria counts were more than 108 cfu g
1 of cheese. Whereas the recommended portion of this product is 30 g (Brasil, 2003), the cheeses made with only one microorganism showed Lb. acidophilus La5 and Bifidobacterium Bb12 counts of 1.28  1010 and 2.38  1010 cfu per 30 g one day after processing, respectively. On the other hand, the cheese containing both microorganisms showed counts of 1.14  1010 cfu of Lb. acidophilus La5, and 1.25  1010 Bifidobacterium Bb12 per 30 g of cheese. This may be an advantage from a technological point of view, since most existing probiotic products contain only one or a
et al., 2013). In the present study, it was few strains (Foligne possible to produce Prato cheese, a semi-hard and mediummoisture cheese, with high counts of lactic acid bacteria and bifidobacteria, which are safe microorganisms for application in food processing.

K.S. Chaves, M.L. Gigante / International Dairy Journal 52 (2016) 10e18

13

Table 1 Physicochemical composition and viability of microorganisms in control and probiotic cheeses after 1 day of refrigerated storage.a Parameter

Control Prato cheese

Composition pH Acidity (%) Fat (%) FDB (%) Moisture (%) Salt (%) S/M (%) Total protein (%) Viability (cfu g
1) Lactococcus lactis sp. Lb. acidophilus La5 Bifidobacterium Bb12 NSLAB

5.06 1.20 28.8 50.9 43.4 2.15 4.72 22.6

± ± ± ± ± ± ± ±

0.02 0.06 0.4 2.4 0.6 0.11 0.28 1.5

3.89  109 <1 <1 1.91  102

Prato cheese with La5

Prato cheese with Bb12

Prato cheese with La5 and Bb12

P-value

5.02 ± 0.04 1.38 ± 0.06 28.3 ± 0.4 49.4 ± 1.6 43.79 ± 0.10 2.20 ± 0.29 4.8 ± 0.6 23.07 ± 0.21

5.15 ± 0.05 1.17 ± 0.04 28.8 ± 0.4 50.7 ± 2.5 42.7 ± 1.5 2.23 ± 0.24 5.0 ± 0.7 23.40 ± 0.9

5.10 1.25 28.9 50.3 42.6 2.03 4.55 24.9

± ± ± ± ± ± ± ±

0.11 0.16 3.0 3.0 2.2 0.08 0.06 2.4

0.768 0.331 0.959 0.867 0.853 0.719 0.872 0.483

2.45  109 4.27  108 <1 7.59  102

1.62  109 <1 7.94  108b 6.03  102

2.24 3.80 4.17 1.48

   

109 108 108b 102

e e e e

a Abbreviations are: FDB, fat on dry basis; S/M, salt in moisture; NSLAB, non-starter lactic acid bacteria. Total protein is calculated as total N  6.38. Values are given as mean ± standard deviation (n ¼ 3, except where indicated). b n ¼ 2.

With respect to the typical characteristics of Prato cheese, it was observed that the addition of Lb. acidophilus La5 and Bifidobacterium Bb12 during the manufacturing process, together or separately, did not affect the physicochemical composition of the cheeses when compared with the control (Table 1). After one day of refrigerated storage, both the control and the probiotic cheeses met the standards of identity and quality for Prato cheese (Brasil, 1997) regarding the moisture content (36.0e45.9%) and fat on a dry basis (45.0e59.9%). When compared with other studies, the cheeses showed higher salt content than the usual range of 1.6e1.9%, and pH values lower than the expected range of 5.2e5.4 (Alves, Merheb-Dini, Gomes, Da Silva, & Gigante, 2013; Cichoscki, Valduga, Valduga, Tornadijo, & Fresno, 2002; Mazal et al., 2007; Vianna, Mazal, Santos, Bolini, & Gigante, 2008). The lowest pH values observed in the cheeses of the present study may be due to the differences in the manufacturing process, since the cheeses produced by Alves et al. (2013), Mazal et al. (2007) and Vianna et al. (2008) were washed-curd cheeses, whereas the cheeses produced in this study were not subjected to the washing step. In the Brazilian industry, Prato cheese can be produced with or without the curd washing process. The protocol without curd washing was selected for this study due to preliminary tests suggesting greater retention of probiotic microorganisms than the washed curd (data not shown). The lower pH range from 5.02 to 5.15 may have favoured salt absorption, since salt diffusion is pH-dependent, thus the lower the pH, the greater the salt absorption (Guinee & Fox, 2004). 3.3. Cheese characterisation during ripening Moisture, pH, acidity, proteolysis [pH 4.6-SN (% TN) and 12% TCA-SN (% TN)], and the concentrations of acetic acid and lactic acid were significantly affected in the control cheese and probiotic cheese (Table 2). The moisture content of the control Prato cheese,

cheese with La5, with Bb12, and with both La5 and Bb12 was 43.4 ± 0.6, 43.79 ± 0.10, 42.7 ± 1.5 and 42.6 ± 2.2%, respectively. Despite the cheeses with La5 (43.79% moisture) and Bb12 (42.7% moisture) being significantly different, no significant differences were observed for the probiotic cheeses when compared with the control. There was no significant difference in the acidity of cheeses with La5 (1.46% lactic acid) and with both probiotics (1.43% lactic acid). These cheeses had higher acidity and differed significantly from the control cheese (1.27% lactic acid) and Bb12 (1.32% lactic acid). Regardless of treatment, the acidity of the cheeses increased significantly during storage. This increase can be due to the consumption of the residual lactose by the cultures, and greater retention of residual lactose in the product, since the process does not include the curd washing step. Faion et al. (2013) observed an increased acidity in the Prato cheese, whose manufacturing process did not include curd washing, as in this study. The authors attributed this increase to the degradation of residual lactose by lactic cultures. Although different acidity values were observed for the control and probiotic cheeses, this same behaviour was not observed for the pH, possibly due to the buffering power of proteins. Despite significant differences, the pH range during ripening between cheeses was 0.07 pH units, and the control cheese (pH 5.02), the cheese with La5 (pH 5.1) and the cheese with La5 and Bb12 (pH 5.5) did not differ. All cheeses containing probiotics showed higher primary proteolysis [pH 4.6-SN (% TN)] than the control cheese (11.7 ± 1.8% TN) with no significant differences (14.9 ± 2.7, 14.7 ± 2.3 and 15.5 ± 1.7% TN) for Prato cheese with La5, Bb12, and La5 plus Bb12, respectively. With respect to the secondary proteolysis [12% TCA-SN (% TN)], cheeses containing Lb. acidophilus La5 as a single strain (7.9 ± 1.2% TN) or in combination with Bb12 (8.1 ± 1.4% TN) showed higher proteolysis, and were different from both control (5.7 ± 1.0%

Table 2 Summary of analysis of variance on the effect of treatments, storage time and interaction between treatment  time on the characteristics of Prato cheeses. Factor

Treatment (T)a Storage time (ST)b T  ST a b

P e value

df

3 5 15

Moisture

pH

Acidity (% lactic acid)

pH 4.6 SN (% TN)

12% TCA SN (% TN)

Firmness (N)

Lactic acid (mg g
1)

Acetic acid (mg g
1)

0.022 0.525 0.995

<0.0001 0.462 1.000

<0.0001 <0.0001 0.878

<0.0001 <0.0001 0.492

<0.0001 <0.0001 0.317

0.195 <0.0001 0.899

<0.0001 0.053 0.120

<0.0001 0.056 0.747

Treatment: control Prato cheese, Prato cheese with La5, Prato cheese with Bb12 and Prato cheese with La5 and Bb12. Storage time: 1, 7, 14, 28, 40 and 60 days after manufacture.

14

K.S. Chaves, M.L. Gigante / International Dairy Journal 52 (2016) 10e18

TN) and cheese containing only Bifidobacterium Bb12 (6.8 ± 1.3% TN). The most intense primary and secondary proteolysis observed in cheeses with Lb. acidophilus may be due to the production of intracellular amino, di and tripeptidases by Lb. acidophilus strains (Shihata & Shah, 2000; Upadhyay, McSweeney, Magboul, & Fox, 2004). Secondary proteolysis of the cheeses containing only Bifidobacterium Bb12 did not differ from the control cheese, probably due to the weak proteolytic activity of the Bifidobacterium strain (Bergamini et al., 2009; Boylston, Vinderola, Ghoddusi, & Reinheimer, 2004; Corbo et al., 2001). Bergamini et al. (2009) found that the addition of Lb. acidophilus alone or in combination with other probiotic cultures contributed to higher secondary proteolysis in probiotic Cheddar, suggesting a greater contribution of this microorganism in the evolution of proteolysis. Regardless of the type of cheese (control and probiotic), storage time affected proteolysis and firmness of cheeses (Table 2), which showed an increase in nitrogen fractions and loss of firmness (Fig. 1). No significant interaction was observed between treatments and storage time for all the nitrogen fractions evaluated [pH 4.6-SN (% TN) and 12% TCA-SN (% TN)] (Table 2). Proteolysis behaviour is dependent upon the action of the residual coagulant, microbial proteinases and peptidases that promote hydrolysis of the protein matrix with reduced firmness and hydration of the protein network (Upadhyay et al., 2004). The hydrolysis profile of the different casein fractions of control and probiotic cheeses was assessed by capillary electrophoresis (Fig. 2) during the 60 days of storage. It was observed that the profile of hydrolysis of casein fractions was similar in all cheeses. However, the migration time of the cheese casein fractions were different for the peaks in Figs. A and B obtained before 50 min and in Figs. C and D before 40 min. This difference may be due to capillary substitution and to the use of new buffer solutions (Recio, Amigo, Ramos, & ~ o, 1997). On the first day of storage, the formation of Lopez-Fandin para-k-casein, aS1-1-CN 8P and aS1-1-CN 9P was observed, which are degradation products of k-casein, aS1-CN 8P and aS1-CN 9P, respectively. After 28 days of storage, the fractions aS1-CN 8P and aS1-CN 9P were hydrolysed in almost all cheeses, indicating the action of residual coagulant on aS1-CN with formation of peptides aS1-1-CN 8P and aS1-1-CN 9P (Fig. 2). The genetic variants b-CN A1 and A2 suffered poor hydrolysis during storage with the formation of small peaks of g-CN, and the formation of the peak g2-CN in the

Fig. 1. Effect of storage time on pH 4.6 SN (% TN; ), 12% TCA SN (% TN; ) and firmness ( ) of Prato cheeses with or without the probiotic microorganisms (n ¼ 3). Lowercase letters indicate values are statistically different by Tukey's test (P < 0.05). SN ¼ soluble nitrogen; TN ¼ total nitrogen; TCA ¼ trichloroacetic acid.

first 7 days of storage. After this period, the fraction b-CN remained almost intact throughout storage, suggesting a low activity of plasmin (Fox, Guinee, Cogan, & McSweeney, 2000) and low specificity of bacterial peptidases on the fraction of b-CN (Ong, Henriksson, & Shah, 2007). The treatments significantly affected the concentration of lactic acid and acetic acid of the cheeses (Table 2). The concentration of lactic acid in cheese with Bb12 was significantly lower when compared with other cheeses. Although cheese with La5 and cheese containing both probiotics were significantly different, no differences were observed when compared with the control cheese. The acetic acid concentration in the cheeses with Bb12 alone or in combination with La5 was not significantly different and was higher than the acetic acid levels found in both the control cheese and cheese with La5 (Table 3). The higher acetic acid and lower lactic acid concentrations in the cheese with Bifidobacterium Bb12 can be due to the metabolism of the bacteria that ferment lactose via fructose-6-phosphate, producing 3 mol of acetic acid and 2 mol of lactic acid (Ballongue, 2004). Further studies are needed to evaluate the effect of acetic acid concentration on the sensory characteristics of Prato cheese containing Bifidobacterium. 3.4. Viability of microorganisms during refrigerated storage of cheeses As observed in Table 4, after 60 days of storage, the Lb. acidophilus La5 population reduced 2.06 and 1.65 log units in the cheeses with these bacteria alone, or in combination with Bifidobacterium Bb12, respectively. The population of Bifidobacterium Bb12 alone or in combination with Lb. acidophilus La5 showed reductions of 1.72 and 1.40 log units, respectively. Despite these losses, after 60 days of storage, the concentration of viable cells ranged from 108 to 109 cfu in 30 g of the product. The viable cells count was 1.12  108 cfu in 30 g of Prato cheese with La5, and 4.53  108 cfu in 30 g of Prato cheese with Bb12. For the cheese containing both probiotics, the viable cells count was 2.55  108 cfu in 30 g of cheese with Lb. acidophilus La5 and 3.48  108 cfu in 30 g of cheese with Bifidobacterium Bb12. There is a consensus today for some well-known strains, such as Lactobacillus and Bifibobacterium, that the functional dose for food or supplements usage should be of the order of 1  109 cfu per portion, as the level established by Health Canada, or a daily intake of 1  109 cfu, as established by the Italian Ministry of Health (Hill et al., 2014). Even though the cheeses obtained at this study met the Brazilian legislation for probiotic food products (bacteria count higher than 108 cfu g
1; Brasil, 2008), they would not meet the legislation of countries such as Italy and Canada. The regulatory framework for probiotics or functional foods in a wider context is
et al., 2013) and still different in distinct parts of the world (Foligne there is a need for harmonisation of scientific developments and regulatory processes (Sanders et al., 2013). Furthermore, the presence of the probiotic microorganisms did not affect the viability of Lactococcus lactis sp. and NSLAB in Prato cheese during 60 days of storage (Table 4). Both the lactococci and NSLAB populations in control and probiotic cheese showed a classical behaviour, that is, a reduction in lactococci viability and an increase in NSLAB populations (Fox et al., 2000). The lower population of lactococci in the cheeses, which was on average 1.06 logarithmic cycles, can be due to intrinsic factors such as pH, salt concentration and lack of
et al., 2007; Fox et al., fermentable carbohydrates (Briggiler-Marco 2000; Ong & Shah, 2009; Vinderola et al., 2000). Regarding NSLAB, the average increase in cheese was 2.6 log units, which can be related to the capacity of some strains to survive heat treatment of milk, the major source of these microorganisms (Beresford & Williams, 2004).

K.S. Chaves, M.L. Gigante / International Dairy Journal 52 (2016) 10e18

15

Fig. 2. Capillary electrophoresis curves showing casein hydrolysis during storage of Prato cheese: (A) control Prato cheese; (B) Prato cheese with La5; (C) Prato cheese with Bb12;
n, and Ardo € (2010). AU ¼ arbitrary units. (D) Prato cheese with La5 and Bb12. Labelling of peaks is according to Alves et al. (2013), Otte et al. (1997), Rehn, Petersen, Saede

Table 3 Effect of treatments on the organic acid concentration (mg g
1) of control and probiotic Prato cheeses.a Acid Lactic acid Acetic acid a

Control Prato cheese ab

14.8 ± 0.6 0.66 ± 0.25b

Prato cheese with La5 a

15.3 ± 0.5 1.0 ± 0.4b

Prato cheese with Bb12 c

12.0 ± 0.6 1.5 ± 0.6a

Prato cheese with La5 and Bb12 13.6 ± 0.6b 1.5 ± 0.4a

Values are means ± standard deviation; different superscript letters in the same row show significant difference by Tukey's test (P < 0.05).

3.5. Simulation of gastrointestinal conditions Tables 5 and 6 show the viability of Lb. acidophilus and Bifidobacterium from Prato cheeses during exposure to artificial gastrointestinal juice, after 28 and 60 days of refrigerated storage. For all probiotic cheeses, the viability of Lb. acidophilus and Bifidobacterium was not affected by treatments, exposure time, or interaction of these factors after 28 and 60 days of storage when exposed to simulated gastrointestinal. The maintenance of viability may be associated with the protective effect of the protein-fat matrix, since the numbers of viable bacteria that reach the duodenum is strongly affected by the protective capacity of the fat from the meal in stomach. (Mainville, Arcand, & Farnworth, 2005; Tompkins, Mainville, & Arcand, 2011). Another factor that may have contributed to microorganism stability may be the increase in pH of the

gastric solution during the experiment, since the protocol did not provide pH adjustments after the cheeses were subjected to artificial gastric juice. In this perspective, it is possible that the methodology used in this study may have underestimated the viability loss of probiotics when compared with the new consensus static in vitro method recently published (Minekus et al., 2014). With regard to exposure to bile after 28 days of storage, the viability of Lb. acidophilus and Bifidobacterium was affected only by the exposure time (Table 7). However, no significant difference in the viability of both microorganisms was observed from the beginning (0 min) to the end of bile exposure (300 min). The treatments and the interaction between treatment and exposure time did not affect the viability of probiotics. After 60 days of storage, the cheeses were exposed to bile to assess the viability of probiotics. The viability of Lactobacillus was not affected by the

16

K.S. Chaves, M.L. Gigante / International Dairy Journal 52 (2016) 10e18

Table 4 Viability of microorganisms in Prato cheese during storage.a Treatment

Storage time (d)

Viability (log cfu g
1) Lc. lactis sp.

Control Prato cheese

Prato cheese with La5

Prato cheese with Bb12

Prato cheese with La5 and Bb12

a b

1 7 14 28 40 60 1 7 14 28 40 60 1 7 14 28 40 60 1 7 14 28 40 60

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.58 9.56 9.36 8.73 8.82 7.86 9.39 9.40 9.38 9.18 9.13 8.90 9.24 9.33 9.32 8.73 8.91 8.39 9.38 9.36 9.36 9.31 9.06 8.17

0.15 0.18 0.13 0.42 0.14 0.36 0.05 0.04 0.31 0.26 0.08 0.31 0.12 0.10 0.22 0.51 0.19 0.33 0.07 0.05 0.19 0.42 0.48 1.01

Lb. acidophilus La5

Bifidobacterium Bb12

NSLAB

<1 <1 <1 <1 <1 <1 8.63 8.38 8.38 8.25 7.54 6.57 <1 <1 <1 <1 <1 <1 8.58 8.36 8.32 8.21 8.26 6.93

<1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 8.90 8.00 8.60 8.02 7.36 7.18 8.62 7.82 8.36 7.80 7.70 7.22

2.28 2.30 2.87 3.15 2.65 5.06 2.88 2.18 2.79 3.29 3.64 4.61 2.78 2.81 3.63 4.11 4.39 6.04 2.17 2.43 3.61 4.43 4.87 4.80

± ± ± ± ± ±

± ± ± ± ± ±

0.37 0.42 0.49 0.40 0.21 0.55

0.93 0.48 0.55 0.55 0.74 0.14

± ± ± ± ± ± ± ± ± ± ± ±

0.62b 0.69b 0.24b 0.27 0.47 0.56 0.30b 0.74b 0.21b 0.44 0.46 0.58

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.66 0.75 0.12 0.33 0.95 0.57 1.09 0.68 0.27 0.75 0.88 0.64 1.03 0.30 1.54 0.46 0.95 0.69 0.15 0.24 0.40 0.45 1.17 0.57

NSLAB, non-starter lactic acid bacteria. Values are means ± standard deviation (n ¼ 3, unless otherwise indicated). n ¼ 2.

Table 5 Viability of Lactobacillus acidophilus La5 during sequential exposure to artificial gastric juice (AGJ) for 2 h and artificial intestinal juice (AIJ) for 300 min after 28 and 60 days of refrigerated storage of the probiotic cheese.a Sequential incubation

Time (min)

28 d storage

60 d storage

Prato cheese with La5 AGJ (pH 2.0)

AIJ (pH 7.0)

0 60 120 300

6.3 6.42 6.7 6.7

± ± ± ±

ns NS

0.4 0.07 0.7 0.5

Prato cheese with La5 and Bb12 6.2 6.2 6.08 6.7

± ± ± ±

0.6 0.5 0.09 0.5

Prato cheese with La5 4.64 5.08 3.8 4.9

± ± ± ±

ns NS

0.08 0.25 1.7 1.1

Prato cheese with La5 and Bb12 4.4 4.6 4.1 4.6

± ± ± ±

1.0 1.4 1.1 1.1

Values (log10 cfu g
1) are given as mean ± standard deviation(n ¼ 3); ns, NS: not significant in column and row, respectively, by F-test for each storage time (28 and 60 days of storage) (P < 0.05). a

Table 6 Viability of Bifidobacterium Bb12 during sequential exposure to artificial gastric juice (AGJ) for 2 h and artificial intestinal juice (AIJ) for 300 min after 28 and 60 days of refrigerated storage of the probiotic cheese.a Sequential incubation

Time (min)

28 d storage

AGJ (pH 2.0)

0 60 120 300

6.30 6.0 5.8 6.7

60 d storage

Prato cheese with La5

AIJ (pH 7.0)

± ± ± ±

0.25 1.8 2.5 1.3

ns NS

Prato cheese with La5 and Bb12 5.6 6.3 5.4 6.5

± ± ± ±

0.4 0.5 2.2 1.5

Prato cheese with La5 4.9 5.8 5.9 6.6

± ± ± ±

0.6 1.3 1.6 1.4

ns NS

Prato cheese with La5 and Bb12 4.9 4.7 5.7 5.9

± ± ± ±

0.7 1.7 1.2 1.4

Values (log10 cfu g
1) are given as mean ± standard deviation(n ¼ 3); ns, NS: not significant in column and row, respectively, by F-test for each storage time (28 and 60 days of storage) (P < 0.05). a

Table 7 Effect of time of exposure to 0.5% bile on the viability of Lactobacillus acidophilus La5 and Bifidobacterium Bb12 in probiotic Prato cheeses after 28 days of refrigerated storage.a Time (min)

L. acidophilus La5 (log cfu g
1)

Bifidobacterium Bb12 (log cfu g
1)

0 60 300

5.29b 6.28a 5.77ab

4.89b 6.62a 5.83ab

a Different superscript letters in the same column over time of exposure show significant difference by Tukey's test (P < 0.05; n ¼ 3).

treatments, exposure time, and interaction between these factors. These results suggest that the probiotic bile resistance is related to the stability of the culture for 60 days of refrigerated storage of the cheeses. The pH of the cheese possibly caused minor damage to the cells when compared with fruit juice, and pH values below 4.5 are considered detrimental to stability of probiotic bacteria during storage, leading to a high loss of viability during GI simulation (Champagne & Gardner, 2008; Sheehan, Ross, & Fitzgerald, 2007). Regarding the bifidobacteria, only the treatments affected their viability, and higher viability (5.70 log cfu g
1) was observed for Prato cheese with Bb12 when compared with cheese with La5 and

K.S. Chaves, M.L. Gigante / International Dairy Journal 52 (2016) 10e18

Bb12 (4.24 log cfu g
1), suggesting that not only the product characteristics and storage time have influenced the stability of probiotic, but also that the resistance is strain-dependent. 4. Conclusions Prato cheese has been shown to be a carrier of probiotic cultures, promoting the continued viability of the microorganisms during the 60 days of refrigerated storage. Furthermore, probiotics confer protection during simulation of the passage through the gastrointestinal tract. The addition of Lb. acidophilus La5 and Bifidobacterium Bb12, separately or in combination did not change the physicochemical composition of the cheeses and their behaviour during ripening. Acknowledgements The authors are grateful to the Foundation for Research Support ~o Paulo (FAPESP) for the financial support of the of the State of Sa project 2012/15160-0 and Coordination of Improvement of Higher Education Personnel (CAPES) for granting the scholarship. References ~ oAbadía-García, L., Cardador, A., Del Campo, S. T. M., Arvízu, S. M., Castan
lez, C., et al. (2013). Influence of probiotic strains Tostado, E., Regalado-Gonza added to cottage cheese on generation of potentially antioxidant peptides, antilisterial activity, and survival of probiotic microorganisms in simulated gastrointestinal conditions. International Dairy Journal, 33, 191e197. Akin, M. B., Akin, M. S., & Kirmaci, Z. (2007). Effects of inulin and sugar levels on the viability of yogurt and probiotic bacteria and the physical and sensory characteristics in probiotic ice-cream. Food Chemistry, 104, 93e99. Albenzio, M., Santillo, A., Caroprese, M., Ruggieri, D., Napolitano, F., & Sevi, A. (2013). Physicochemical properties of Scamorza ewe milk cheese manufactured with different probiotic cultures. Journal of Dairy Science, 96, 2781e2791. Alves, L. S., Merheb-Dini, C., Gomes, E., Da Silva, R., & 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. (2006a). Direct forced air oven drying, method no. 990.19. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th ed., p. 35). Gaithersburg, MD, USA: AOAC International. AOAC. (2006b). Gerber method, method no. 2000.18. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th ed., pp. 21e23). Gaithersburg, MD, USA: AOAC International. AOAC. (2006c). Gravimetric method, method no. 945.46. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th ed., p. 10). Gaithersburg, MD, USA: AOAC International. AOAC. (2006d). Kjeldahl method, method no. 2001.14. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th ed., pp. 72e73). Gaithersburg, MD, USA: AOAC International. AOAC. (2006e). Kjeldahl method, method no. 991.20. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th ed., pp. 10e12). Gaithersburg, MD, USA: AOAC International. AOAC. (2006f). Method I, method no. 926.08. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th ed., pp. 68e69). Gaithersburg, MD, USA: AOAC International. AOAC. (2006g). Method V, method no. 979.13. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th ed., pp. 37e38). Gaithersburg, MD, USA: AOAC International. AOAC. (2006h). Titrimetric method, method no. 920.124. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th ed., p. 73). Gaithersburg, MD, USA: AOAC International. Ballongue, J. (2004). Bifidobacteria and probiotic action. In S. Salminem, A. von Wright, & A. Ouwehand (Eds.), Lactic acid bacteria e Microbiological and functional aspects (pp. 67e124). New York, NY, USA: Marcel Dekker, Inc. Beresford, T., & Williams, A. (2004). The microbiology of cheese ripening. In P. F. Fox, P. L. H. McSweeney, T. M. Cogan, & T. P. Guinee (Eds.), General aspects: Vol. 1. Cheese: Chemistry, physics and microbiology (pp. 287e317). London, UK: Elsevier Applied Science. Bergamini, C. V., Hynes, E. R., Palma, S. B., Sabbag, N. G., & Zalazar, C. A. (2009). Proteolytic activity of three probiotic strain in a semi-hard cheese as single and mixed cultures: Lactobacillus acidophilus, Lactobacillus paracasei and Bifidobacterium lactis. International Dairy Journal, 19, 467e475. Bergamini, C. V., Hynes, E. R., & Zalazar, C. A. (2006). Influence of probiotic bacteria on the proteolysis profile of a semi-hard cheese. International Dairy Journal, 16, 856e866.

17

Blaiotta, G., La Gatta, B., Di Capua, M., Di Luccia, A., Coppola, R., & Aponte, M. (2013). Effect of chestnut extract and chestnut fiber on viability of potential probiotic Lactobacillus strains under gastrointestinal tract conditions. Food Microbiology, 36, 161e169. Boylston, T. D., Vinderola, C. G., Ghoddusi, H. B., & Reinheimer, J. A. (2004). Incorporation of bifidobacteria into cheeses: challenges and rewards. International Dairy Journal, 14, 375e387.
ria e Abastecimento e MAPA. Portaria n Brasil. (1997). Minist
erio da Agricultura Pecua
cnico para 358, de 04 de setembro de 1997, que aprova o Regulamento Te ~o de Identidade e Qualidade de Queijo Prato. Dia
rio Oficial da Unia ~o: Fixaça ~o 1. (Ministry of Brasília, Distrito Federal, em 08 de setembro de 1997. Seça Agriculture, Livestock and Food Supply - MAPA. Ordinance No. 358, September 4, 1997. Technical Rules for Identity and Quality of Prato Cheese. Official Gazette: Brasilia, Federal District, September 08, 1997. Section 1.). ^ncia Sanita
ria - ANVISA. Resoluça ~o RDC nº Brasil. (2003). Ag^ encia Nacional de Vigila
cnico de Porço ~es de Ali359, de 23 de dezembro de 2003. Regulamento Te ~o: mentos Embalados para Fins de Rotulagem Nutricional. Di
ario Oficial da Unia Brasília, Distrito Federal, em 26 de dezembro de 2003. (National Health Surveillance Agency - ANVISA. Resolution RDC No. 359 of 23 December 2003. Regulation of Packaged Food Portions for Nutritional Labelling. Official Gazette: Brasilia, Federal District, December 26, 2003.). Available at http://www.anvisa. gov.br. ^ncia Sanita
ria e ANVISA. Alimentos com Brasil. (2008). Ag^ encia Nacional de Vigila ~es de propriedades funcionais e ou de saúde, novos alimentos/ingrealegaço
ticos: lista de alegaço ~es de propriedade dientes, subst^ ancias bioativas e probio funcional aprovadas. Atualizado em julho de 2008. (National Health Surveillance Agency - ANVISA. Foods with functional and or health claims, new foods/ ingredients, bioactive compounds and probiotics: list of approved functional property claims.). Available at http://www.anvisa.gov.br.
ria e Abastecimento e MAPA e Brasil. (2011). Minist
erio da Agricultura Pecua ~o Normativa n 62, de 29 de setembro de 2011. que altera a IN 51 de 2002 Instruça
cnicos de Produça ~o, Identidade e Qualidade do e aprova os Regulamentos Te Leite tipo A, do Leite Cru Refrigerado, do Leite Pasteurizado e o Regulamento
cnico da Coleta de Leite Cru Refrigerado e seu Transporte a Granel. Dia
rio Te Oficial da Uni~ ao: Brasília, Distrito Federal, em 29 de dezembro de 2011. Seç~ ao 1. (Ministry of Agriculture, Livestock and Supply - MAPA - Normative Ruling No. 62, 29 September 2011 amending IN 51, 2002 and approving the Technical Regulations for Production, Identity and Quality of Cooled Raw Milk, Pasteurized Milk and the Technical Regulation of Cooled Raw Milk and Transport in Bulk tanks. Official Gazette: Brasilia, Federal District, December 29, 2011. Section 1.).
, M., Capra, M. L., Quiberoni, A., Vinderola, G., Reinheimer, J. A., & Briggiler-Marco Hynes, E. (2007). Nonstarter Lactobacillus strains as adjunct cultures for cheese making: In vitro characterization and performance in two model cheeses. Journal of Dairy Science, 90, 4532e4542. BSI.. (1989). Determination of fat content of milk and milk products (Gerber methods). Methods (p. 12). London, UK: British Standard Institution. Buriti, F. C. A., Rocha, J. S., & Saad, S. M. I. (2005). Incorporation of Lactobacillus acidophilus in Minas fresh cheese and its implications for textural and sensorial properties during storage. International Dairy Journal, 15, 1279e1288. Bynum, D. G., & Barbano, D. M. (1985). Whole milk reverse osmosis retentate for Cheddar cheese manufacture: chemical changes during aging. Journal of Dairy Science, 68, 1e10. Champagne, C. P., & Gardner, N. J. (2008). Effect of storage in a fruit drink on subsequent survival of probiotic lactobacilli to gastro-intestinal stresses. Food Research International, 41, 539e543. 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, 329e336. Corbo, M. R., Albenzio, M., De Angelis, M., Sevi, A., & Gobbetti, M. (2001). Microbiological and biochemical properties of Canestrato Pugliese hard cheese supplemented with bifidobacteria. Journal of Dairy Science, 84, 551e561.
langer, G., & Vuillemard, J. C. (1999). Production of probiotic Daigle, A., Roy, D., Be cheese (Cheddar-like cheese) using enriched cream fermented by Bifidobacterium infantis. Journal of Dairy Science, 82, 1081e1091. Darukaradhya, J., Phillips, M., & Kailasapathy, K. (2006). Selective enumeration of Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria and non-starter lactic acid bacteria from Cheddar cheese. International Dairy Journal, 16, 439e445. Donkor, O. N., Nilmini, S. L. I., Stolic, P., Vasiljevic, T., & Shah, N. P. (2007). Survival and activity of selected probiotic organisms in set-type yoghurt during cold storage. International Dairy Journal, 17, 657e665. Faion, A. M., Beal, P., Ril, F. T., Cichoski, A. J., Cansian, R. L., Valduga, A. T., et al. (2013). Influence of the addition of natural antioxidant from mate leaves (Ilex paraguariensis St. Hill) on the chemical, microbiological and sensory characteristics of different formulations of Prato cheese. Food Science and Technology, 52, 1516e1524.
, B., Daniel, C., & Pot, B. (2013). Probiotics from research to market: the Foligne possibilities, risks and challenges. Current Opinion in Microbiology, 16, 284e292. Fox, P. F., Guinee, T. P., Cogan, T. M., & McSweeney, P. L. H. (2000). Fundamentals of cheese science. Gaithersburg, MD, USA: Aspen Publishers, Inc. Gerez, C. L., Font de Valdez, G., Gigante, M. L., & Grosso, C. R. F. (2012). Whey protein coating bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low pH. Letters in Applied Microbiology, 54, 552e556.

18

K.S. Chaves, M.L. Gigante / International Dairy Journal 52 (2016) 10e18

Gomes, A. M. P., Vieira, M. M., & Malcata, F. X. (1998). Survival of probiotic microbial in a cheese matrix during ripening: simulation of rates of salt diffusion and microorganism survival. Journal of Food Engineering, 36, 281e301. Guinee, T. P., & Fox, P. F. (2004). Salt in cheese: physics, chemistry, and biological aspects. In P. F. Fox, P. L. H. McSweeney, T. M. Cogan, & T. P. Guinee (Eds.), General aspects: Vol. 1. Cheese: Chemistry, physics and microbiology (pp. 207e259). London, UK: Elsevier Applied Science. Hernandez-Hernandez, O., Muthaiyan, A., Moreno, F. J., Montilla, A., Sanz, M. L., & Ricke, S. C. (2012). Effect of prebiotic carbohydrates on the growth and tolerance of Lactobacillus. Food Microbiology, 30, 355e361. Hill, C., Guarner, F., Reid, G., Gibson, G. R., Merenstein, D. J., Pot, B., et al. (2014). Expert consensus document: the International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews. Gastroenterology and Hepatology, 11, 506e514.
nez-Flores, R. (2002). Rapid simultaneous determinaIzco, J. M., Tormo, M., & Jime tion of organic acids, free amino acids, and lactose in cheese by capillary electrophoresis. Journal of Dairy Science, 85, 2122e2129. Karimi, R., Mortazavian, A. M., & Da Cruz, A. G. (2011). Viability of probiotic microorganisms in cheese during production and storage: a review. Dairy Science and Technology, 91, 283e308.
ria. M.A. M
LANARA. (1981). Secretaria de Defesa Agropecua etodos Analíticos Oficiais de Controle de Produtos de origem animal e seus Ingredientes. II-M
etodos Físicos e
rio da agricultura, Pecua
ria e Abastecimento. Químicos. Brasília-DF: Ministe Secretariat of Agriculture Defense. Official Methods for Examination of Animal Products. II-Physical and Chemical Methods. Brasilia-DF: Ministry of Agriculture, Livestock and Supply. Lavermicocca, P., Valerio, F., Lonigro, S. L., De Angelis, M., Morelli, L., Callegari, M. L., et al. (2005). Study of adhesion and survival of Lactobacilli and Bifidobacteria on table olives with the aim of formulating a new probiotic food. Applied and Environmental Microbiology, 71, 4233e4240. Liu, Z., Jiang, Z., Zhou, K., Li, P., Liu, G., & Zhang, B. (2007). Screening of bifidobacteria with acquired tolerance to human gastrointestinal tract. Anaerobe, 13, 215e219. Mainville, I., Arcand, Y., & Farnworth, E. R. (2005). A dynamic model that simulates the human upper gastrointestinal tract for the study of probiotics. International Journal of Food Microbiology, 99, 287e296. Marsili, R. T., Ostapenko, H., Simmons, R. E., & Green, D. E. (1981). High performance liquid chromatographic determination of organic acids in dairy products. Journal of Food Science, 46, 52e57. Mazal, G., Vianna, P. C. B., Santos, M. V., & Gigante, M. L. (2007). Effect of somatic cell count on Prato cheese composition. Journal of Dairy Science, 90, 630e636. Mc Brearty, S., Ross, R. P., Fitzgerald, G. F., Collins, J. K., Wallace, J. M., & Stanton, C. (2001). Influence of two commercially available bifidobacteria cultures on cheddar cheese quality. International Dairy Journal, 11, 599e610. Milesi, M. M., McSweeney, P. L. H., & Hynes, E. R. (2008). Viability and contribution to proteolysis of an adjunct culture of Lactobacillus plantarum in two model cheese systems: cheddar cheese-type and soft-cheese type. Journal of Applied Microbiology, 105, 884e892. Minekus, M., Alminger, M., Alvito, P., Balance, S., Bohn, T., Bourlieu, C., et al. (2014). A standardised static in vitro digestion method suitable for food - an international consensus. Food and Function, 5, 1113e1124. Mozzi, F., Gerbino, E., Font de Valdez, G., & Torino, M. I. (2009). Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system. Journal of Applied Microbiology, 107, 56e64. Oberg, C. J., Moyes, L. V., Domek, M. J., Brothersen, C., & McMahon, D. J. (2011). Survival of probiotic adjunct cultures in cheese and challenges in their enumeration using selective media. Journal of Dairy Science, 94, 2220e2230. Oliveira, M. N., Sodini, I., Remeuf, F., & Corrieu, G. (2001). Effect of milk supplementation and culture composition on acidification, textural properties and microbiological stability of fermented milks containing probiotic bacteria. International Dairy Journal, 11, 935e942. Ong, L., Henriksson, A., & Shah, N. P. (2006). Development of probiotic Cheddar cheese containing Lactobacillus acidophilus, Lb. casei, Lb. paracasei and Bifidobacteriums spp. and the influence of these bacteria on proteolytic patterns and production of organic acid. International Dairy Journal, 16, 446e456. Ong, L., Henriksson, A., & Shah, N. P. (2007). Proteolytic pattern and organic acid profiles of probiotic Cheddar cheese as influenced by probiotic strains of Lactobacillus acidophilus, Lb. paracasei, Lb. casei or Bifidobacterium sp. International Dairy Journal, 17, 67e78.

Ong, L., & Shah, N. P. (2009). Probiotic Cheddar cheese: Influence of ripening temperatures on survival of probiotic microorganisms, cheese composition and organic acid profiles. Food Science and Technology, 42, 1260e1268. Ortega, N., Albillos, S. M., & Busto, M. D. (2003). Application of factorial design and response surface methodology to the analysis of bovine caseins by capillary zone electrophoresis. Food Control, 14, 307e315. Otte, J., Zakora, M., Kristiansen, K. R., & Qvist, K. B. (1997). Analysis of bovine caseins and primary hydrolysis products in cheese by capillary zone electrophoresis. Lait, 77, 241e257. € Ozer, B., Uzun, Y. S., & Kirmaci, H. A. (2008). Effect of microencapsulation on viability of Lactobacillus acidophilus LA5 and Bifidobacterium bifidum BB12 during Kasar cheese ripening. International Journal of Dairy Technology, 61, 237e244. Picot, A., & Lacroix, C. (2004). Encapsulation of bifidobacteria in whey protein based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. International Dairy Journal, 14, 505e515. Possemiers, S., Marzorati, M., Verstraete, W., & Van de Wiele, T. (2010). Bacteria and chocolate: a successful combination for probiotic delivery. International Journal of Food Microbiology, 141, 97e103. ~ o, R. (1997). Application of capillary Recio, I., Amigo, L., Ramos, M., & Lopez-Fandin electrophoresis to the study of proteolysis of caseins. Journal of Dairy Research, 64, 221e230.
n, K. H., & Ardo €, Y. (2010). Ripening of extra-hard Rehn, U., Petersen, M. A., Saede cheese made with mesophilic DL-starter. International Dairy Journal, 20, 844e851. Richardson, G. H. (1985). Standard methods for examination of dairy products. Washington, DC, USA: American Public Health Association. Ross, R. P., Fitzgerald, G., Collins, K., & Stanton, C. (2002). Cheese delivering biocultures -probiotic cheese. Australian Journal of Dairy Technology, 57, 71e78. Sanders, M. E., Guarner, F., Guerrant, R., Holt, P. R., Quigley, E. M. M., Sartor, R. B., et al. (2013). An update on the use and investigation of probiotics in health and disease. Gut, 62, 787e796. Scheller, M., & O'Sullivan, D. J. (2011). Comparative analysis of an intestinal strain of Bifidobacterium longum and a strain Bifidobacterium animalis subspecies lactis in Cheddar cheese. Journal of Dairy Science, 94, 1122e1131. Schillinger, U., Guigas, C., & Holzapfel, W. H. (2005). In vitro adherence and other properties of lactobacilli used in probiotic yoghurt-like products. International Dairy Journal, 15, 1289e1297. Sheehan, V. M., Ross, P., & Fitzgerald, G. F. (2007). Assessing the acid tolerance and the technological robustness of probiotic cultures for fortification in fruit juices. Innovative Food Science and Emerging Technologies, 8, 279e284. Shihata, A., & Shah, N. P. (2000). Proteolytic profiles of yogurt and probiotic bacteria. International Dairy Journal, 10, 401e408. Shimakava, Y., Matsubara, S., Yuki, N., Ikeda, M., & Ishikawa, F. (2003). Evaluation of Bifidobacterium breve strain Yakult-fermented soymilk as a probiotic food. International Journal of Food Microbiology, 81, 131e136. Stanton, C., Gardiner, G., Lynch, P. B., Collins, J. K., Fitzgerald, G., & Ross, R. P. (1998). Probiotic cheese. International Dairy Journal, 8, 491e496. Terzaghi, B. E., & Sandine, W. E. (1975). Improved medium for lactic streptococci and their bacteriophages. Applied Microbiology, 29, 807e813. Tompkins, T. A., Mainville, I., & Arcand, Y. (2011). The impact of meals on a probiotic during transit through a model of the human upper gastrointestinal tract. Beneficial Microbes, 2, 295e303. Tripathi, M. K., & Giri, S. K. (2014). Probiotic functional foods: survival of probiotics during processing and storage. Journal of Function Foods, 9, 225e241. Upadhyay, V. K., McSweeney, P. L. H., Magboul, A. A. A., & Fox, P. F. (2004). Proteolysis in cheese during ripening. In P. F. Fox, P. L. H. McSweeney, T. M. Cogan, & T. P. Guinee (Eds.), General aspects: Vol. 1. Cheese: Chemistry, physics and microbiology (pp. 391e433). London, UK: Elsevier Applied Science. Vianna, P. C. B., Mazal, G., Santos, M. V., Bolini, H. M. A., & Gigante, M. L. (2008). Microbial and sensory changes throughout the ripening of Prato cheese made from milk with different levels of somatic cells. Journal of Dairy Science, 91, 1743e1750. Vinderola, C. G., Prosello, W., Ghiberto, D., & Reinheimer, J. A. (2000). Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and non probiotic microflora in Argentinian fresco cheese. Journal of Dairy Science, 83, 1905e1911. Vinderola, C. G., & Reinheimer, J. A. (2000). Enumeration of Lactobacillus casei in the presence of L. acidophilus, bifidobacteria and lactic starter bacteria in fermented dairy products. International Dairy Journal, 10, 271e275.