Monday, 25 June 2018

The addition of inulin and Lactobacillus casei 01 in sheep milk ice cream

Food Chemistry Volume 246, 25 April 2018, Pages 464-472 Food Chemistry Author links open overlay panelCelso F.BalthazaraHugo L.A.SilvaaErick A.EsmerinoaRamon S.RochabJeremiasMoraesbMariana A.V.CarmocLucianaAzevedocIhosvanyCampsdYuriK.D AbudeCelsoSant'AnnaeRobson M.FrancoaMônica Q.FreitasaMarcia C.SilvabRenata S.L.RaicesbGraziela B.EscherfDanielGranatofC.Senaka RanadheeragFilomenaNazarrohAdriano G.Cruzb a Universidade Federal Fluminense (UFF), Faculdade de Veterinária, 24230-340, Niterói, RJ, Brazil b Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Departamento de Alimentos, 20270-021, Rio de Janeiro, RJ, Brazil c Universidade Federal de Alfenas (UNIFAL), Faculdade de Nutrição, 37130-000, Alfenas, MG, Brazil d Universidade Federal de Alfenas (UNIFAL), Departamento de Física, 37133-840, Alfenas, MG, Brazil e Instituto Nacional de Metrologia, Normalização e Qualidade Industrial (Inmetro), Duque de Caxias, 25250-020, Rio de Janeiro, Brazil f Universidade Estadual de Ponta Grossa (UEPG), Departamento de Engenharia de Alimentos, 84030-900, Ponta Grossa, Brazil g University of Melbourne, Faculty of Veterinary & Agricultural Sciences, School of Agriculture & Food, Melbourne, VIC 3010, Australia h Istituto di Scienze dell'Alimentazione, CNR-ISA, Via Roma, 64, 83100 Avellino, Italy Received 21 August 2017, Revised 28 November 2017, Accepted 4 December 2017, Available online 5 December 2017. crossmark-logo Get rights and content Referred to by Celso F. Balthazar, Hugo L.A. Silva, Erick A. Esmerino, Ramon S. Rocha, Jeremias Moraes, Mariana A.V. Carmo, Luciana Azevedo, Ihosvany Camps, Yuri K.D. Abud, Celso Sant'Anna, Robson M. Franco, Mônica Q. Freitas, Marcia C. Silva, Renata S.L. Raices, Graziela B. Escher, Daniel Granato, C. Senaka Ranadheera, Filomena Nazzaro, Adriano G. Cruz Corrigendum to “The addition of inulin and Lactobacillus casei 01 in sheep milk ice cream” [Food Chem. 246 (2018) 464–472] Food Chemistry, Volume 252, 30 June 2018, Pages 397 Download PDF Highlights • Inulin and L. casei addition in sheep milk ice cream formulation. • Inulin addition did not improve probiotic counts and CaCo-2 adhesion. • Inulin improved the ACE inhibitory and antioxidant activity and hardness. • Probiotic and synbiotic sheep milk ice creams presented different volatiles compounds and improved acid organic levels. Abstract The effect of the Lactobacillus casei 01 and inulin addition on sheep milk ice cream during storage (−18 °C, 150 days) was investigated. Control, probiotic and synbiotic ice cream (10% w/w sheep milk cream; 10% w/w sheep milk cream, L. casei 01, 6 log CFU/mL; 10% w/w inulin, L. casei 01, 6 log CFU/mL, respectively) were manufactured. Microbiological counts (probiotic count, survival after in vitro gastrointestinal resistance, Caco-2 cell adhesion), bioactivity and microstructure were analysed. Physical and textural characteristics, colour parameters, thermal analysis and organic acids/volatile compounds were also evaluated. All formulations supported L. casei 01 viability and maintained above the minimum therapeutic level (>6 log CFU/mL) during storage. Inulin did not affect L. casei 01 survival after the passage through simulated gastrointestinal tract and adhesion to Caco-2 cells while improved the ACE-inhibitory and antioxidant activity. L. casei 01 addition produced several volatile compounds, such as carboxylic acids, alcohols, aldehydes and ketones. Also, scanning electron microscopy showed an interaction between probiotic bacteria and inulin fibre on synbiotic ice cream and the adhesion of L. casei to Caco-2 cells was observed. Previous article Next article Keywords L. casei 01 Ice cream Sheep milk Bioactivity Caco 2 adhesion Functional foods 1. Introduction Probiotics are defined as live microorganisms which when administered in adequate amounts, confer a health benefit on the host (Hill et al., 2014). The probiotic effects are strain-specific (Espitia, Batista, Azeredo, & Otoni, 2016) and they should possess resistant to the gastric and bile acids, adhere to mucus or human enteric epithelial cells, antimicrobial activity against pathogenic bacteria, bile salt hydrolase activity, and ability to reduce pathogen adhesion to surfaces of gastrointestinal tract (Kolaček et al., 2017). Prebiotics are “substrate that is selectively utilized by host microorganisms conferring a health benefit” (Gibson et al., 2017). The prebiotic fibres consist of inulin and other oligosaccharides. Thus, the term synbiotic refers to the synergistic effect between prebiotic foods and selective probiotic microorganisms (Cencic & Chingwaru 2010). Many animal and human studies have demonstrated that the consumption of products containing both probiotics and prebiotics can provide health benefits, improving the survivability and deployment of probiotics in the food supplements into the gastrointestinal tract by promoting the selective growth and/or activating bacteria metabolism (Miremadi, Sherkat, & Stojanovska, 2016). The wide use of inulin in the food industry is based on its technological attributes and the great interest to develop healthy products aiming at the consumers’ requirement. These products include fibre-enriched, prebiotic, low fat and low sugar foods (Ahmed & Rashid, 2017). As supplement in skim dairy products, inulin considerably increases the growth and sustainability of Lactobacillus spp. and Bifidobacterium spp. in non-fat fermented milk (Closa-Monasterolo et al., 2013), including L. casei-01 (Paseephol & Sherkat, 2009). Various prebiotic dairy desserts with low fat content have been prepared using inulin as a prebiotic, in which inulin supplementation not only was giving a prebiotic effect but also was reducing the fat content and sugar content without affecting its sensory acceptability (Arcia et al., 2011, Balthazar et al., 2015a, Balthazar et al., 2017b). Milk products such as ice cream and frozen desserts may serve as carriers for delivering the probiotics and prebiotics to the human gut (Ayar, Siçramaz, Öztürk, & Yilmaz, 2017). The high total solids level in ice cream including the fat and milk solids can protect the probiotic bacteria. In addition, the production and storage of ice cream have relatively little effect on the probiotic survival when compared to fermented milk products, once the probiotic bacteria are able to survive under freezing temperatures for long periods. However, the cell membrane of the probiotic bacteria is damaged by the freezing process, leading to injuries that compromise the cell function and metabolic activity (Tripathi & Giri, 2014). In this context, considering that ice cream is widely appreciated worldwide, the supplementation with prebiotic ingredients and/or probiotic bacteria can add value to the product by providing a functional appeal. In addition, studies have shown that bacterial cultures remain on ice cream at levels sufficient to offer the suggested therapeutic effects (Cruz, Antunes, Sousa, Faria, & Saad, 2009). Sheep milk is an interesting raw material for the production of ice creams, as it is rich in nutrients and contains high total solids levels (Balthazar et al., 2017a). No additional protein or fat rather than sheep milk cream is needed, as well not requiring homogenization during the manufacturing process (Balthazar et al., 2017b, Balthazar et al., 2017c). Therefore, this study aimed at investigating the effect of the addition of Lactobacillus casei 01 and inulin to sheep milk ice cream. The microbiological determinations (probiotic bacteria counts, survival through simulated conditions of the gastrointestinal tract, and Caco-2 cell adhesion), bioactivity (ACE and DPPH values), and metabolic profiling (volatile compounds and organic acids), as well as the microstructure, physical, textural and colour parameters of sheep milk ice cream were evaluated. 2. Material and methods 2.1. Ice cream processing Three different sheep milk ice creams were manufactured: a conventional full-fat sheep milk ice cream (CONV); a probiotic full-fat sheep milk ice cream (PROB), and a synbiotic non-fat sheep milk ice cream (SYNB). Whole raw sheep milk containing 5% (v/v) of fat (about 13%, w/v, non-fat solids) was obtained from a herd of Lacaune sheep located in the mountainous region of Rio de Janeiro, Brazil. Sheep milk was skimmed to 0.1% (w/w) of fat and sheep milk cream containing 64% (w/v) fat was collected. Finally both of skimmed milk and cream were heat-treated (72–75 °C/15 s) using pasteurizer plates (BCISMINI, EQUILATI, São Paulo, Brazil). The processing of milk and derived products was carried out at a dairy laboratory pilot plant (Instituto GPA – Núcleo Avançado de Educação em Tecnologia de Alimentos e Gestão de Cooperativismo, NATA, São Gonçalo/RJ, Brazil). As per the manufacturer’s recommendation, a 100 mg (w/w) of freeze dried Lactobacillus casei-01 (about 6 log CFU/g, Chr Hansen, Valinhos, SP, Brazil) in each 1 L (v/v) of skimmed sheep milk (w/v) was separately added to probiotic and synbiotic non-fat fermented sheep milk preparation. Fermentation was carried out at 37 ± 1 °C and reached pH 4.7–4.6 in 6 h. Subsequently, samples were stored at 5 ± 2 °C in 1 L polypropylene containers. Sheep milk ice creams were manufactured in accordance with standard protocols. To prepare 2 L of ice cream mix of each treatment, 1.69 L of skimmed sheep milk was added to 0.31 L of sheep milk cream (10% w/w fat) in CONV; 0.69 L of skimmed sheep milk was added to 0.31 L of sheep milk cream (10% w/w fat) in PROB; and 1 L of skimmed sheep milk was added to 200 g of inulin (10% w/w) in SYNB. To make 2 L of ice cream mix, non-fat fermented sheep milk was added (1 L) in PROB and SYNB treatments. The following ingredients were added to each mix: 20 g of stabilizer and emulsifier (Selecta®, Two Wheels, Jaragua do Sul, Brazil), 300 g sucrose (União, Refined, São Paulo, SP, Brazil) and 2 mL vanilla essence (Arcolor®, São Paulo, SP, Brazil). The mixes were heated to 50 °C to dissolve the ingredients and vanilla flavouring was added after cool down the mixture temperature (5 °C ± 2). Ice cream mixes were maturated for 24 h in a domestic refrigerator (5 °C ± 2), then submitted to air incorporation (45%, 3 min, −5 °C, Arpifrio Pro 9, Santo André, SP, Brazil). Finally, sheep milk ice creams were packaged in propylene containers (v = 200 mL) and stored at −18 °C during 150 days. The experiment was carried out three times. 2.2. Microbiological analysis 2.2.1. L. casei 01 counts and survival after gastrointestinal resistance The counts of L. casei 01 on ice cream, after survival through simulated gastrointestinal tract and the in vitro adhesion to Caco-2 cells were performed in triplicates during frozen storage. The enumeration was performed using Man, Rogosa, and Sharpe agar (MRS Agar, HiMedia Laboratories, Mumbai, India) added with Vancomycin. Vancomycin stock solution (2% w/v) was prepared with distilled water and filter-sterilized using a 0.45 µm membrane (EMD Millipore, Merck KGaA, Darmstadt, Germany) and 0.5 mL solution was added into 1 L base medium at the time of analysis. Plates were incubated at 37 °C ± 1 °C for 72 h under anaerobiosis condition. The count was expressed as the number of colony-forming units per gram of ice cream (log CFU/mL). The survival after in vitro gastrointestinal tract condition was evaluated considering previous study (Felicio et al., 2016) with modifications. During the gastric phase, the pH was adjusted to 2.0–2.3 using sterile 0.5 mol/L HCl. Sterile pepsin (Sigma-Aldrich, St. Louis, MO, USA) and lipase (Amano lipase F-AP15; Aldrich Chemical Company, Milwaukee, WI, USA) solutions were added to samples to reach concentrations of 3 g/L and 0.9 mg/L, respectively. In the enteric phase, the pH was raised to 7.0–8.0 using a sterile alkaline solution containing bile and pancreatin at the concentrations of 10 and 1 g/L, respectively. The Petri plate counts were performed in triplicate during frozen storage (1st, 90th and 150th day) and after survival in vitro gastrointestinal tract simulation (1st, 90th and 150th day). 2.2.2. In vitro adhesion assay Coloretal adenocarcinoma epithelial cells (Caco-2) was obtained from Rio de Janeiro cell Bank (BCRJ) (Rio de Janeiro, Brazil). Caco-2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (Sigma-Aldrich, USA) supplemented with 20% fetal bovine serum (FBS, Gibco, São Paulo, Brazil) and 100 IU penicillin/100 µg streptomycin (Sigma-Aldrich, USA). The cell line was grown in a 37 °C humidified incubator containing 5% CO2. Caco-2 cells were plated into 24-well plates at a density of 105 cells/well and the cell line was incubated in humidified atmosphere at 37 °C in 5% CO2 until a confluent monolayer had formed (7–10 days). One day before the adhesion assay, the growth medium was replaced with the same medium without antibiotics (penicillin/streptomycin) and washed three times with sterile phosphate buffered saline (PBS). A 1 mL aliquot of the ice cream was transferred to post-confluent monolayers of Caco-2 cells and incubated at 37 °C in 5% CO2 for 2 h in the humidified incubator. The remaining food particles were subsequently removed and the cells were washed thoroughly with PBS in order to remove non adherent Lactobacillus. Caco-2 was detached from each well by addition of 1 mL of trypsin/EDTA (Sigma Aldrich, USA) and the suspension (1 mL) from each well was then transferred to a tube containing 9 mL of peptone water, serially diluted. A 1 mL was transferred to plates containing MRD medium and the Lactobacillus were grown in anaerobiosis at 37 °C for 72 h. The assay was carried out in triplicates in order to determine adhesion ability during frozen storage (1st, 90th and 150th day). Adhesion percentages were calculated by determining the viable bacterial counts of the ice cream before the adhesion assay and viable bacterial counts adhered to the Caco-2 cells. 2.2.3. Atomic force microscopy (AFM) Caco-2 monolayer and Lactobacillus casei-01 images were obtained from AFM for the qualitative examination of probiotic adhesion into Caco-2 cells. The 256 × 256 pixel resolution AFM images were obtained with a Park NX10 microscope using the True Non-Contac™ Mode. The initial scan size was 50 × 50 µm made with a NSC15 cantilever (from MikroMasch) at 0.15 Hz scan rate, 3.5 Z servo gain and 6 nm set point. 2.2.4. Scanning electron microscopy (SEM) The SEM was carried out according to Matias, Padilha, Bedani, and Saad (2016), to check the interaction between probiotic bacteria and inulin fibre (SYNB) and probiotic with ice cream structures (PROB). Briefly, the samples, obtained from probiotic and synbiotic ice creams at storage were centrifuged (5000g for 10 min) to remove the supernatant. The resulting pellets were resuspended in NaCl solution (0.9%, w/v) at a final concentration of around 5 log CFU/mL. The cell suspensions (1 mL) were filtered through 0.2 mm-pore size membrane filters (Isopore, Millipore, Billerica, MA, USA) and fixed in a 2% (w/v) glutaraldehyde solution for 2 h. Afterwards, the membranes were washed using Milli-Q water (Millipore). The washing procedure was repeated two more times, after which the membranes were dehydrated in ethanol solutions in the following sequence: 25, 50, 75, 90, and 95%, and finally with 100% ethanol (three times), and dried using the critical-point CO2 method. The dried membranes were placed on aluminium stubs, sputter coated with gold and their analyses were performed using a field emission scanning electron microscope (JEOL JSM-7401F; JEOL, Tokyo, Japan) at 2.5 kV. Sample images were acquired at different magnifications (×5000, ×15,000 and ×30,000). 2.3. Physical and colour analysis 2.3.1. pH The pH value was determined using a digital potentiometer (Model PG1800, Chapter Lab®, SP, Brazil). The pH measurement was performed in triplicates at the moment of probiotic bacteria counts were performed. 2.3.2. Overrun To determine the overrun (%) of sheep milk ice cream, a known mass of mix (mmix) and ice cream (mice cream) sample were weighted and the percentage of overrun was calculated according to Eq. (1) (Rinaldi et al., 2013). (1) 2.3.3. Melting The melting behaviour, expressed as melting rate (%), was evaluated as described by Di Criscio et al. (2010). Briefly, 80 ± 8 g of ice cream (−18 °C) were placed on a wire mesh screen (4 Tyler mesh) and left to melt into a 250 mL Becker at room temperature (24 ± 2 °C) until 50% of the sample was melted. The weight of the melted ice cream was recorded every 5 min aiming at obtaining a sigmoidal curve representing the kinetics of the melting process. From the linear part of the curve, the most probable straight line was calculated, in which its slope represented the melting rate (g/min). 2.3.4. Fat destabilization Fat destabilization ratio (%) was evaluated on the first day of storage as described by Balthazar et al. (2017b). Briefly, samples were diluted at 1:500 (v/v) with distilled water, centrifuged for 5 min at 450g and, 10 min later, the absorbance of the solution was measured at 540 nm using a spectrophotometer (model Bio2000, Bioplus, Barueri/SP, Brazil) against a blank (distilled water). The absorbance of the diluted mix was compared to that of the diluted melted ice cream. Fat destabilization was calculated as Eq. (2). (2) 2.3.5. Colour parameters Instrumental colour measurement was performed at −18 °C one day after the manufacture of ice creams using a portable colorimeter (CR-410, Konica Minolta Sensing, Inc., Tokyo, Japan). The coordinates L∗, a∗ and b∗ were obtained using the CIE system, where L∗ is a measure of the lightness, a∗ varies from green (−) to red (+) and b∗ varies from blue (−) to yellow (+) using D65 illuminate and observer at 10°. The chroma (C∗) was calculated using Eq. (3) and whiteness index (WI) was calculated using Eq. (4) (Balthazar et al., 2015a), respectively. (3) (4) 2.4. Thermal analysis Differential scanning calorimetry (DSC) were obtained using a Perkin–Elmer calorimeter (Diamond, Perkin-Elmer, Norwalk, PA) and Pyris software for Windows. The DSC instrument was calibrated with pure standard indium before analysis (Balthazar et al., 2017b). Aliquots (15 mg) of each sample (solution or ice cream mixture) were sealed into aluminium pans (50 µL, Perkin-Elmer) and placed into the DSC equipment. A protocol similar to that proposed by Kavaz, Yuksel, and Dagdemir (2015) was implemented including the following steps: (1) holding 1.0 min at −60 °C, (2) heating from −60 °C to −40 °C at 10 °C/min, (3) cooling from −40 °C to −60 °C at 10 °C/min, (4) holding for 5 min at −60 °C; (5) heating from −60 °C to 20 °C at 5 °C/min; (6) annealing at the same temperature for 30 min to promote maximal ice formation; (7) cooling to 80 °C at 10 °C/min and holding at 80 °C for 5 min and (8) heating from 80 °C to 20 °C at 5 °C/min. Data collected for sheep milk ice cream included the glass transition temperature (Tg) and specific heat (ΔCP), the onset (Tm) and peak melting temperatures, and ice fusion latent enthalpy (ΔH). The unfreezable water (UFW), according to Alvarez, Wolters, Vodovotz, and Ji (2005), was calculated by subtracting the sheep milk ice cream moisture (%) from freezable water (%) at the peak temperature. Effective molecular weight (Ms) was determined according to the method proposed by Soukoulis and Tzia (2018): (5) where Tf, T0 the freezing point temperature as it was determined by DSC and the freezing point temperature of pure water, respectively (in K), Xw is the mass fraction of water, Xb is the mass fraction of bound water, Xs is the mass fraction of dry matter, Mw is the molecular weight of pure water (kg/mol), Lf is the ice fusion latent heat (J/kg) and R is the perfect gas constant. Ice fusion latent heat was calculated using the formula: (6) where T is the temperature expressed in °C. 2.5. Textural parameters Apparent viscosity was evaluated after 24 h of aging at 4 °C using Q860M21 rotational viscometer microprocessor (Quimis®, São Paulo, SP, Brazil). The #2 spindle at 10 rpm was used and the readings were recorded (mPa.s) at the best instrument accuracy according to the manual's instructions, which was around 50% FSR (full-scale range) (Rinaldi et al., 2013). Hardness was evaluated using the TA-XT2i texture analyzer (Stable Micro Systems, Godalming, UK), calibrated with a 25 kg load cell. The ice cream samples were maintained at − 18 °C until the analysis. During testing, the samples were compressed by penetration of a 2 mm diameter aluminium cylinder probe (36R). The tests were carried out in the compression power mode with a pre-test speed of 2 mm/s, and test and post-test speeds of 1 mm/s, with a penetration distance of 10 mm. Three determinations were performed at approximately −18 °C for each batch. Hardness was measured as the peak compression force (N) during probe penetration, and stickiness was determined as the negative peak force (N) during withdrawal (Balthazar et al., 2017c). 2.6. Bioactivity parameters The angiotensin I-converting enzyme inhibitory (ACEI) activity was determined following the spectrophotometric assay (Torres-Lianez, González-Córdova, Hernandez-Mendoza, Garcia, & Vallejo-Cordoba, 2011). The extent of inhibition was calculated as follow, being A the absorbance in the presence of angiotensin I-converting enzyme (ACE) and the ACEI component, B the absorbance without the ACEI component, and C the absorbance without ACE: (7) The antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging method (Lee, Jeewanthi, Park, & Paik, 2016). The DPPH radical-scavenging activity was calculated using the following formula: (8) 2.7. Volatile organic compounds The volatile organic compounds analysis was performed as previously described (Condurso, Verzera, Romeo, Ziino, & Conte, 2008) with modifications. The volatile organic compounds were extracted by solid phase micro extraction (SPME) and analyzed by gas chromatography (Agilent Technologies® 6890A gas chromatograph) coupled to mass spectrometry (Agilent Technologies® 5973 mass spectrometer). The SPME extractions were carried out using 50/30 mm thick DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) fibres (Supelco, Bellefonte, PA, USA) and 20 mL headspace vials in an manual sampler holder (Supelco, Bellefonte, PA, USA). For that, 3 g sample was added with 3 mL saturated NaCl solution maintaining the vial at 40 °C in a dry block (Multi-Blok 2003 Thermo Fisher Scientific, Germany) with a 20 min equilibration time and 30 min extraction time. After extraction, the SPME fibre was manually introduced into the GC–MS for 30 min under the following conditions for the thermal desorption of the analytes: injector temperature at 240 °C; splitless injection mode; column CP-Wax 52 CB 60 m, 0.25 mm id, 0.25 mm film thickness; oven temperature programmed from 45 °C for 5 min, then increased to 80 °C at a rate of 10 °C min−1, then to 240 °C at 5 °C min−1 and kept for 30 min; 1 mL min−1 of Helium gas flow, transfer line temperature at 240 °C, electron impact ionization energy at 70 eV, acquisition mass range of 50–550 m/z. The volatile compounds were identified by comparison of their experimental spectra with those provided by the National Institute of Standards & Technology (NIST/EPA/NIH Mass Spectra Library, NIST11, USA), using the linear retention indices (LRI). LRI values of C8-C40 alkanes standards (Supelco, 40,127-U) were injected under the same chromatographic and mass spectrometric conditions. 2.8. Organic acids The organic acids levels (lactic, acetic and citric) were quantified as described by Felicio et al. (2016) using high performance liquid chromatography (HPLC) (Ultimate 3000 Thermo Fisher Scientific, Gremering, Germany) using an Aminex X-87H column (300 mm × 7.8 mm × 9 µm, Bio-Rad Laboratories, Richmond, CA, USA) and a guard column containing disposable H+ cartridges (Biorad-Rad Laboratories) at 65 °C. The mobile phase was sulfuric acid (0.009 mol/L), previously diluted in ultra-pure water (Milli-Q system, Millipore Corporation, Billerica, MA, USA), and subsequently filtered and degassed through a 0.45 µm membrane filter (Millipore). The flow rate was 0.6 mL/min, and UV–Vis detection was performed at 220 nm, with volume injection of 25 µL. Standards of lactic, acetic, and citric acids were prepared, and the chromatographic peaks were integrated using the Chromeleon 6.8 software (Ultimate 3000 Thermo Fisher Scientific, Gremering, Germany). 2.9. Statistical analyses The experiment was repeated three times and all analyses were performed in triplicate. Instrumental data were analysed using one-way analysis of variance (ANOVA) and the comparison of means was conducted using the Fisher’s LSD test at 5% confidence level (Granato, de Araújo Calado, & Jarvis, 2014). The statistical analyses were conducted using the XLSTAT software version 2017.1 (Adinsoft, Paris, France). 3. Results and discussion 3.1. Microbiological analysis The L. casei 01 viability and survival after simulated passage through the gastrointestinal tract, adhesion to Caco-2 cell in vitro, and percentage of adhesion were assessed in probiotic full-fat ice cream (PROB), synbiotic non-fat ice cream (SYNB), and its respective ice cream mixes during storage (−18 °C) as well pH values of sheep milk ice creams are shown in Table 1. The initial probiotic counts in both sheep milk ice cream mixes were 7.9 log CFU/g, probably because of the similar fermentation procedure, as described in ice cream manufacture section. In contrast, a reduction of 0.74 log cycle (p < .05) was observed for the bacteria viability in the PROB ice cream after overrun (day 1), with no significant reductions in SYNB ice cream, with value of 0.04 log cycle. A greater viability of the probiotic strain was observed in the SYNB ice cream (7.06 log CFU/g), after passage through the simulated conditions of gastrointestinal tract (p < .05) when compared to the PROB ice cream (5.72 log UFC/g) containing the same probiotic strain. However, a significant (p < .05) reduction of bacteria counts was observed in both ice creams at day 150, with values of 5.02 and 5.18 log CFU/g for PROB and SYNB, respectively. At day 1 of storage, from the initial viable cells (7.16 and 7.86 log CFU/g in PROB and SYNB, respectively), 5.69 and 5.70 log CFU/g were capable to adhere to Caco-2 cells, representing an adhesion capacity of 79% and 73% for PROB and SYNB, respectively (Table 1), which reduced significantly (p < .05) to 5.18 (75%) and 5.09 log CFU/g (67%), respectively, at day 150 of storage. The significant differences (p < .05) in pH values of sheep milk ice creams were basically because of the type of sheep milk used to manufacture the ice creams. This observation was expected because the conventional sheep milk ice cream (CONV) was made from sheep milk only, while the probiotic ice creams contained 50% fermented milk in their composition. In addition, the significant differences (p < .05) in acidity of probiotic and synbiotic ice creams were probably due to the addition of inulin in SYNB ice cream. Table 1. Values of pH, viability in mix and ice cream, survival through gastrointestinal simulation assay and adhesion to CaCO-2 cells of Lactobacillus casei 01 in sheep milk ice creams.1 Sheep milk ice cream Day** pH Viability in mix Viability in ice cream Survival** Adhesion** % CONV 1 6.82a ± 0.02 – – – – 90 6.80a ± 0.02 – – – – 150 6.81a ± 0.02 – – – – PROB 1 5.30c ± 0.10 7.90a ± 0.10 7.16b ± 0.10 5.72b ± 0.01 5.69a ± 0.21 79 90 5.33c ± 0.06 – 7.00b ± 0.10 5.10c ± 0.17 5.24b ± 0.09 75 150 5.30c ± 0.10 – 6.89b ± 0.06 5.02c ± 0.12 5.18b ± 0.07 75 SYNB 1 5.70b ± 0.10 7.90a ± 0.10 7.86a ± 0.10 7.06a ± 0.01 5.70a ± 0.01 73 90 5.67b ± 0.06 – 7.70a ± 0.10 5.26c ± 0.24 5.12b ± 0.01 66 150 5.67b ± 0.06 – 7.61a ± 0.10 5.18c ± 0.06 5.09b ± 0.01 67 *Values expressed in mean ± standard deviation. a–c Different letters at the same column mean significant difference (p < .05) between treatments. **It was noted effect along during days of cold storage. Viability in mix and ice cream and survival through gastrointestinal resistance are expressed in log CFU/mL. CaCO-2 adhesion is expressed in log CFU/mL and percentage of adhesion (%). 1 Sheep milk ice creams containing: 10% v/v sheep milk cream (CONV); 10% v/v sheep milk cream and L. casei-01 (PROB); and 10% w/v inulin and L. casei-01 (SYNB). Concerning the probiotics viability after air incorporation and freezing steps, probably inulin exerted a protective effect to the probiotics against oxidation on account of the presence of oxygen and fast temperature drop during freezing of SYNB, which was not observed in PROB, which showed a significant decrease (p < .05) in the viable cell counts. During storage, the viable bacteria counts remained constant and were around 9 log CFU per serving, considering the ice cream serving in many countries including Brazil. Usually, probiotic strains are anaerobic, therefore, oxygen is toxic; and freezing step also injuries bacterial cells, resulting in a decrease in the bacterial count (Pandiyan, Annal Villi, Kumaresan, Murugan, & Gopalakrishnamurthy, 2012). However, no significant decrease in viability was observed during storage, thus inulin was not responsible for maintaining the viability of L. casei-01 during freezing. Di Criscio et al. (2010) reported that storage at −20 °C did not interfere with the viability of L. casei in ice creams, which remained above 7 log. With respect to the probiotic survival after simulated passage through the gastrointestinal tract, no effect of inulin on preventing bacteria death along the tract was observed in SYNB, while L. casei-01 counts reduced almost 2 log cycle. This finding is in-line with a recent study covering soy and/or milk-based synbiotic apple ice creams in which the probiotic counts were also improved after the simultaneous addition of milk and soy (Matias et al., 2016). This behaviour may be due to injuries in the membrane cell during frozen storage (Tripathi & Giri, 2014). A significant decrease of probiotic viability (5.02 log CFU/g; p < .05) in PROB was also observed. Neither fat nor inulin protected the viable probiotic cells from the injuries under freezing conditions. Fortunately, both sheep milk ice creams can be considered probiotic products, because sufficient bacteria numbers (above 7 log per serving) were delivered to the gut. According to Casarotti, Carneiro, and Penna (2014) and Espitia et al. (2016), probiotics must achieve the intestine locus for colonization in viable cell counts at a minimum dose of 6 log CFU per day to ensure therapeutic effects. Lactobacillus casei 01 and inulin fibre structure in SYNB ice cream were clearly observed in SEM microscopy (Fig. 1). The probiotic cells appear dispersed in the PROB ice cream environment, with normal morphological structure, evidenced by a smooth continuous surface along the cell structure. It is also suggested the inulin acts as a “support point” for the L. casei 01 in the SYNB ice cream. Download high-res image (200KB)Download full-size image Fig. 1. Scanning electron micrograph of two rod-shaped Lactobacillus casei-01 of probiotic sheep milk ice cream (PROB, A); and adhered rod-shaped Lactobacillus casei-01 into inulin fibre from synbiotic sheep milk ice cream (SYNB, B). A high adhesion (>50%) of L. casei-01 to Caco-2 cells was observed in PROB and SYNB ice creams (Fig. 2). Our findings are in accordance with other authors, who also found high adhesion of L. casei and L. acidophilus to Caco-2 cells (Adriana et al., 2016, Ranadheera et al., 2012). Certain probiotics may have strong adhesive capacities due to various physiological and biochemical properties, such as the presence of mucus-binding pili on the cell wall that facilitates the adhesion process (Kankainen et al., 2009). Nevertheless, Deepika, Rastall, and Charalampopoulos (2011) also reported a reduction of bacterial adhesion in yogurts and ice creams during storage, because storage time demonstrated a greater influence in maintaining the bacterial adherence when compared to other factors, such as fat and sugar contents of carrier food matrices. Download high-res image (492KB)Download full-size image Fig. 2. Caco-2 monolayer and Lactobacillus casei images obtained from Atomic Force Microscope (AFM, Park NX10). Caco-2 monolayers (A, B). Arrows highlight the cell prominent microvilli aspects that cover part of the cell surfaces forming the intestinal brush border; segment with Caco-2 monolayers completed covered by Lactobacillus casei, without showing the microvilli feature; arrow points out the slightly aspect of Lactobacillus casei (C, D). Lactobacillus casei densely arranged and adhered into Caco-2 monolayers (E). Lactobacillus casei feature organized on plate without Caco-2 monolayers (F). The Caco-2 cell line is used as an in vitro model to screen the adhesive ability of Lactobacillus strains. It is one of the most widely used cell lines in studies about the adherence of probiotics and pathogens to the intestinal epithelium (Abedi, Feizizadeh, Akbari, & Jafarian-Dhkordi 2013). Good adherence of probiotic bacteria to the intestinal cells is related to many health benefits, including the exclusion of enteropathogens (Hashemi, Shahidi, Mortazavi, Milani, & Eshaghi 2014). Probiotics adhesion to epithelial cells of the gastrointestinal tract can modulate the indigenous microbes, enhance the intestinal epithelial barrier, stimulate the host immune system, prevent infections of enteropathogens at an early stage by inhibiting the bacteria and competition for nutrients and attachment sites, or by secreting antimicrobial substances (Lim & Ahn 2012). 3.2. Physical analysis of sheep milk ice cream An increased overrun value (p < .05) was observed in CONV when compared to both PROB and SYNB ice creams (Table 2), while the fat destabilization was significantly higher in CONV (35.77%), followed by PROB (7%), and SYNB (4.63%) ice creams (p < .05). Moreover, both the melting rate and the melting time were significantly (p < .05) lower in PROB when compared to CONV, with values of 1.94 g/min and 50 min, and 2.04 g/min and 55 min for PROB and CONV, respectively, while SYNB ice cream exhibited melting time of 55 min, as observed for CONV. Overrun is an important physical characteristic of ice creams, as it affects the product's quality, interfering with texture, softness, and stability of the product and it is defined as the percent of ice cream expansion on account of the incorporation of air into the ice cream mix during freezing (Dertli et al., 2016). Significant differences in overrun values were observed for sheep milk ice creams, probably because of the type of milk used in the formulation, as fermented skim sheep milk was used in PROB and SYNB ice creams. Possibly the fermented milk structure provided lower overrun in fermented sheep milk ice creams, as also reported by Senanayake, Fernando, Bamunuarachchi, and Arsekularatne (2013). The amount of air incorporated also determines the melting rate, once probiotic ice creams normally exhibit low overrun when compared to non-probiotic ice creams, probably because of the lower melting rate of probiotic ice creams (Senanayake et al., 2013). In addition, the inulin did not affect overrun, with no significant differences when compared to the full-fat sheep milk ice creams (Balthazar et al., 2017b). The fat destabilization had an increased correlation with the overrun values (0.973; p < .05). Destabilized fat in ice cream takes the form of clumps of fat globules that coat and support the air cells (Muse & Hartel, 2004). Table 2. Physical, textural, colour and thermal parameters of sheep milk ice creams.1 Parameters Sheep milk ice creams CONV PROB SYNB Physical Overrun 87.00a ± 0.02 77.50b ± 0.01 78.00b ± 0.01 Melting Rate 2.04a ± 0.00 1.94b ± 0.01 2.00ab ± 0.00 Fat Destabilization. 35.77a ± 0.15 7.00b ± 0.10 4.63c ± 0.84 Textural Apparent Viscosity 61.87c ± 2.22 276.07b ± 5.06 1632.03a ± 24.42 Hardness 46.79b ± 4.42 42.58b ± 2.98 88.01a ± 13.37 Colour L* 93.00a ± 1.46 94.04a ± 0.26 94.84a ± 0.83 C* 13.45a ± 1.05 13.82a ± 0.32 13.57a ± 1.39 WI 84.76a ± 0.32 84.94a ± 0.37 85.48a ± 1.56 Thermal Tg −28.32a ± 0.92 −27.74a ± 1.24 −30.38b ± 0.43 ΔCp 1.01b ± 0.18 1.37b ± 0.47 2.77a ± 0.16 Tm −8.38a ± 0.09 −8.56b ± 0.10 −8.26a ± 0.60 ΔH 108.57b ± 8.71 128.50a ± 5.85 119.11ab ± 1.14 UFW 35.10a ± 1.27 31.59b ± 1.65 37.06a ± 0.90 Ms 490.64b ± 7.21 539.09a ± 7.50 452.25c ± 3.04 *Values expressed in mean ± standard deviation. a-c Different letters at the same row mean significant difference (p < .05). Overrun, fat destabilization and UFW (unfreezable water) are expressed in percentage. Melting rate is expressed in g/min. Apparent viscosity is expressed in mPa.s. Hardness is expressed in N. Colour parameters (L*, C*, WI) are expressed without dimension. Tg (glass transition temperature) and Tm (melting temperature) are expressed in °C. ΔCp (specific heat) is expressed in J/g*°C. ΔH (ice fusion latent enthalpy) is expressed in J/g. Ms (effective molecular weight) is expressed in g/mol. 1 Sheep milk ice creams containing: 10% v/v sheep milk cream (CONV); 10% v/v sheep milk cram and L. casei-01 (PROB); and 10% w/v inulin and L. casei-01 (SYNB). The non-fat ice cream mix (1632.03 mPa.s) had the highest apparent viscosity value (p < .05), followed by PROB (276.07 mPa.s) and CONV (61.87 mPa.s) on account of the use of fermented sheep milk rather than whole sheep milk in the ice cream formulation. With respect to the parameter hardness, the SYNB ice cream (88.01 N) presented a significantly higher value when compared to other sheep milk ice creams (p < .05), reaching twice the value of PROB (42.58 N) and CONV (46.79 N). High apparent viscosity of ice creams containing inulin is expected (Balthazar et al., 2017c, Isik et al., 2011), due to texture improvement provide by this fibre (Shoaib et al., 2016). In this study, the addition of inulin increased the apparent viscosity and hardness of SYNB ice cream. Regards the colour parameters, the brightness values of sheep milk ice creams ranged from 93 (CONV) to 95 (SYNB), with no significant difference between samples (p > .05). Colour is an important attribute in food; it is the first characteristic perceived by the consumers and thus often influences the consumer’s preference (Mani-López, Palou, & López-Malo, 2014). Moreover, whiteness is an important characteristic of sheep milk-based products (Balthazar et al., 2017a, Balthazar et al., 2017b). In the present study, sheep milk ice creams were extremely white-coloured, and inulin and fermented milk did not affect the colour parameters, with no significant differences between ice cream formulations. The thermal analysis indicated differences in most all parameters evaluated (p < .05). Indeed, the glass transition temperature (Tg; −27.74 and −28.32 to −30.38 °C for PROB, CONV and SYNB, respectively), specific heat (ΔCp; 2.77 and 1.01–1.37 J/g∗°C for SYNB, CONV and PROB, respectively), onset-melting temperature (Tm; −8.56 to −8.26 and −8.38 °C for PROB and SYNB, CONV, respectively), ice fusion latent enthalpy (ΔH; 128.5–108.57 J/g for PROB and CONV, respectively), unfreeze water (UFW; 37.06 and 35.1–31.59% for SYNB, CONV, and PROB, respectively) and effective molecular weight (Ms; 539.09 and 490.64 and 452.25 g/mol for PROB, CONV and SYNB, respectively) presented differences (p > .05) among the ice creams formulations, as can be seen in Table 2. An endothermic peak was observed in the DSC thermograms of sheep milk ice creams (data not shown), probably because of ice melting process, as also reported in other studies (Alvarez et al., 2005, Balthazar et al., 2017b). Most differences in calorimetric parameters were observed for PROB and SYNB ice creams, demonstrating that the replacement of fat for inulin had a significant impact on the calorimetric parameters of sheep milk ice creams. Similar result was reported by Balthazar et al. (2017b) in sheep milk ice cream with fat replaced by dietary fibres. However, thermal studies on sheep milk are scarce. The pH values of sheep milk ice creams did not change during cold storage (CONV: 6.82–6.81; PROB: 5.33–5.30; and SYNB: 5.70–5.67; p > .05). Possibly, the lower pH values of PROB ice cream were responsible for the lower melting rate of this sample, because acidity may raise the foam stability, resulting in lower melting (Senanayake et al., 2013). 3.3. Bioactivity of sheep milk ice cream The ACE-inhibitory (ACEI) activity of sheep milk ice creams is presented in Table 3. SYNB (78.4%) exhibited the highest ACEI, followed by PROB (66.67%) and CONV (18.55%) ice creams (p < .05). In addition, the antioxidant activity (% DPPH inhibition) of sheep milk ice cream ranged from 22.73% (CONV) to 81.29% (SYNB, p < .05; Table 3). The scavenging ability of DPPH free radical is widely used to evaluate the antioxidant potential of a particular compound, once antioxidants can neutralize free radicals by accepting or donating electrons (Ullah et al., 2015). The probiotic strain enhanced the bioactive peptides with antioxidant activity, while inulin probably prevented their degradation in the SYNB, once higher DPPH activity (p < .05) were observed in sheep milk ice creams. Table 3. ACE-inhibitory activity (ACEI) and antioxidant activities (DPPH) activities in sheep milk ice creams.1 Sheep milk ice creams ACEI DPPH CONV 18.55c ± 0.66 22.73c ± 0.59 PROB 66.67b ± 1.22 72.16b ± 0.01 SYNB 78.40a ± 0.51 81.29a ± 1.06 *Values expressed in mean ± standard deviation. a-c Different letters at the same column mean significant difference (p < .05). ACE and DPPH are expressed in percentage inhibition (%). 1 Sheep milk ice creams containing: 10% v/v sheep milk cream (CONV); 10% v/v sheep milk cream and L. casei 01 (PROB); and 10% w/v inulin and L. casei-01 (SYNB). Inhibition of ACE is considered a therapeutic approach for hypertension treatment (Miremadi et al., 2016). Bioactive peptides have been identified and characterized in many dairy products such as cheese, yogurt, and fermented milk. ACEI peptides are released from parent protein by the digestive enzymes, microbial and plant enzymes, or proteolytic activities of starter cultures and probiotic bacteria during milk fermentation (Moslehishad et al., 2013). In the present study, it is suggested that the probiotic strain was responsible for the enhancement of ACEI activity in sheep milk ice creams. Thus, probably the ACEI is due to the presence of bioactive peptides released from milk casein during fermentation (Miremadi et al., 2016). Actually, the fraction of β-casein molecule in milk contains amino acid sequences with ACEI effects that are inactive unless released by the proteolytic action of digestive and microbial enzymes (Korhonen, 2009). Indeed, a higher ACEI activity was observed in SYNB when compared to the probiotic ice cream (p < .05), indicating that inulin may have interfered with the probiotic growth and proteolysis, leading to an increase in generation of bioactive peptides, as also reported by Ramchandran and Shah (2010). However, little is known about the effect of inulin on the proteolytic and ACE-inhibitory activities of fermented sheep milk products. 3.4. Metabolites Table 4 shows 41 volatile organic compounds identified in sheep milk ice creams, represented mainly by carboxylic acids (36.58%) followed by alcohols (31.70%). The percentages of carboxylic acids and alcohols, in the ice cream formulations were 45% and 20%; 38.71 and 29.03%; and 44.44 and 25.93% for CONV, PROB, and SYNB, respectively. Some volatile organic compounds have been associated with probiotic metabolism, such as carboxylic acids (2), alcohols (2), aldehyde (1), and ketone (1), which were detected only in the probiotic and synbiotic ice creams. Non-starter lactic acid bacteria, such as L. casei, have an important role in volatile organic compounds production during milk fermentation on dairy products (Dan et al., 2017a), because of their metabolic characteristics and activities (Gobbetti, De Angelis, Di Cagno, Mancini, & Fox, 2015). An example was hexanoic acid that is an important aroma compound frequently presented in different fermented dairy products (Dan et al., 2017b), responsible for goaty flavour and have a lipid source, probably originated from lysine (Peralta, Wolf, Bergamini, Perotti, & Hynes, 2014). A previous study has also found low concentrations of volatile compounds in sheep milk yogurts (Donkor, Nilmini, Stolic, Vasiljevic, & Shah, 2007), because of the oxidation of saturated fatty acids followed by decarboxylation of keto acids (Stefanovic et al., 2017). In this study, other 12 compounds were found in all ice creams, probably owing to the metabolic activities of native microorganisms in raw milk and the ingredients used in sheep milk ice creams formulation. Table 4. Volatile compounds identified in sheep milk ice creams.1 Volatile compound LRI Sheep milk ice creams CONV PROB SYNB Toluol 1024 X Hexanal 1064 X X X 1-Decene 1029 X 2-Heptanone 1164 X X d-limonene 1172 X X Hexanoic acid 1215 X γ-Terpinene 1219 X X 2-Nonanone 1370 X X X 1-Hexanol 1330 X 3-Hexen-1-ol, (Z)- 1348 X X 2-Buten-1-ol, 3-methyl- 1305 X Nonanal 1374 X X 2-Nonanone 1371 X X 1-Hexanol, 2-ethyl- 1466 X X X Ethanol, 2-butoxy- 1381 X 1-Octen-3-ol 1427 X 4-Heptanol, 2,6-dimethyl- 1456 X Benzaldehyde 1506 X X X 1-Octanol 1534 X X 2-Undecanone 1581 X X Ethanol, 2-(2-ethoxyethoxy)- 1595 X X Butanoic acid 1607 X X X Acetophenone 1650 X Pentanoic acid 1669 X 2-Tridecanone 1966 X Cycloheptene 2007 X 3-Decen-1-ol, acetate, (Z)- 2009 X Hexanoic acid 2018 X X Benzyl alcohol 2048 X X X Phenylethyl alcohol 2084 X Heptanoic acid 2124 X Phenol 2175 X X Octanoic acid 2229 X X X Sorbic acid 2314 X X Nonanoic acid 2334 X n-Decanoic acid 2439 X X X Benzoic acid 2697 X X X Dodecanoic acid 2748 X X X Tetradecanoic acid 3018 X X X n-Hexadecanoic acid 3281 X X X Octadecanoic acid 3614 X X 9-Octadecenoic acid – X Oleic Acid – X *LRI = linear retention indices. 1 Sheep milk ice creams containing: 10% v/v sheep milk cream (CONV); 10% v/v sheep milk cream and L. casei-01 (PROB); and 10% w/v inulin and L. casei-01 (SYNB). Regarding the organic acids, higher lactic acid and acetic acid levels were observed in SYNB when compared to PROB (1.55 and 0.94 mg/L against 1.26 and 0.74 mg/L; p < .05, respectively), probably due to higher production of lactic and acetic acids in synbiotic ice creams, which may be related to the interaction between inulin and L. casei 01 during ice cream maturation. Some Lactobacillus strains have heterofermentative behaviour, fermenting glucose to lactic acid and acetic acid. In addition, the inulin can improve the metabolic activity of several lactobacilli species, including L. casei (Donkor et al., 2007). On the other hand, no significant difference was observed for the citric acid levels between PROB and SYNB ice creams. Citric acid is the predominant organic acid in milk (Balthazar et al., 2017a), and its concentration reduces after heat treatment of milk. Therefore, in this study, it can be stated that the amount of citric acid in ice creams remained from sheep milk, suggesting a decrease of citric acid levels in sheep milk after heating, which was not observed in yogurts. 4. Conclusion Sheep milk ice cream has proven to be suitable food matrix for Lactobacillus casei 01 growth and intestinal epithelial cell adhesion in vitro, ensuring therapeutic effect to the host during 150 days of frozen storage (−18 °C). Moreover, Lactobacillus casei 01 presented strong adhesive capacity to Caco-2 cells in vitro. Although the addition of inulin to the synbiotic non-fat ice cream may have improved the potential nutritional benefits, it did not interfere with the survival of L. casei 01 during the passage through simulated gastrointestinal tract or adhesion to Caco-2 cells in vitro. 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