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Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying

Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus... ORIGINAL RESEARCH published: 03 May 2016 doi: 10.3389/fmicb.2016.00584 Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying 1 2 2 3 Natalia Sosa , Esteban Gerbino , Marina A. Golowczyc , Carolina Schebor , 2 4 Andrea Gómez-Zavaglia * and E. Elizabeth Tymczyszyn 1 2 Facultad de Bromatología, Universidad Nacional de Entre Ríos, Gualeguaychú, Argentina, Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA, CCT-CONICET), La Plata, Argentina, Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina, Laboratorio de Microbiología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Argentina In this work maltodextrins were added to commercial galacto-oligosaccharides (GOS) in a Edited by: Michael Gänzle, 1:1 ratio and their thermophysical characteristics were analyzed. GOS:MD solutions were University of Alberta, Canada then used as matrices during spray-drying of Lactobacillus plantarum CIDCA 83114. The Reviewed by: obtained powders were equilibrated at different relative humidities (RH) and stored at 5 Fernanda Mozzi, ◦ ◦ Centro de Referencia para and 20 C for 12 weeks, or at 30 C for 6 weeks. The T s of GOS:MD matrices were about Lactobacilos (CERELA-CONICET), 20–30 C higher than those of GOS at RH within 11 and 52%. A linear relation between Argentina the spin-spin relaxation time (T ) and T-T parameter was observed for GOS:MD matrices Sergio I. Martinez-Monteagudo, g South Dakota State University, USA equilibrated at 11, 22, 33, and 44% RH at 5, 20, and 30 C. Spray-drying of L. plantarum *Correspondence: CIDCA 83114 in GOS:MD matrices allowed the recovery of 93% microorganisms. In Andrea Gómez-Zavaglia contrast, only 64% microorganisms were recovered when no GOS were included in the angoza@qui.uc.pt dehydration medium. Survival of L. plantarum CIDCA 83114 during storage showed Specialty section: the best performance for bacteria stored at 5 C. In a further step, the slopes of the This article was submitted to linear regressions provided information about the rate of microbial inactivation for each Food Microbiology, a section of the journal storage condition (k values). This information can be useful to calculate the shelf-life of Frontiers in Microbiology spray-dried starters stored at different temperatures and RH. Using GOS:MD matrices Received: 08 February 2016 as a dehydration medium enhanced the recovery of L. plantarum CIDCA 83114 after Accepted: 11 April 2016 spray-drying. This strategy allowed for the first time the spray-drying stabilization of a Published: 03 May 2016 potentially probiotic strain in the presence of GOS. Citation: Sosa N, Gerbino E, Golowczyc MA, Keywords: galacto-oligosaccharides, maltodextrin, glass transition temperature, spray-drying, lactic acid bacteria Schebor C, Gómez-Zavaglia A and Tymczyszyn EE (2016) Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Abbreviations: GOS, galacto-oligosaccharides; T , glass transition temperature; MD, maltodextrin; DSC, differential Spray-Drying. Front. Microbiol. 7:584. scanning calorimetry; H-NMR, proton nuclear magnetic resonance; RH, relative humidity; d.b., dry basis; DP, degree of doi: 10.3389/fmicb.2016.00584 polymerization; FID, free induction decay analysis; CFU, cell forming units. Frontiers in Microbiology | www.frontiersin.org 1 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices INTRODUCTION Patel, 2008; Fazaeli et al., 2012). However, the thermophysical properties of some commercial GOS are not the most appropriate Galacto-oligosaccharides (GOS) are non-digestible ones to support their use as dehydration media. Their low T oligosaccharides composed of a variable number of galactose (Tymczyszyn et al., 2012) leads to obtaining sticky products units (usually from 2 to 10) and a terminal glucose unit, linked that agglomerate and adhere to the internal wall of the drying mostly by β1-4 and β1-6 bonds (Casci and Rastall, 2006; Vera chamber, thus resulting in a low product yield and low bacterial et al., 2011). They are widely known because of their prebiotic cultivability (Hennigs et al., 2001; Bhandari and Howes, 2005). properties (Gibson and Roberfroid, 1995) and more recently, To overcome this problem, high molecular weight saccharides they demonstrated to enhance the recovery of lactic acid bacteria of high T , including maltodextrins (MD) or starch, have been during dehydration processes (Tymczyszyn et al., 2011, 2012; generally used to avoid the undesirable stickiness of low T sugar Santos et al., 2014a,b,c). The protective properties of GOS solutions (Bhandari et al., 1997; Fazaeli et al., 2012; Rajam and have been explained on the basis of their ability to form glassy Anandharamakrishnan, 2015). matrices of high viscosity and low molecular mobility where Taking into account that commercial GOS generally include molecular interactions are restricted (Tymczyszyn et al., 2011, different amounts of glucose and lactose, and their purification 2012; Golowczyc et al., 2013; Santos et al., 2014a,b,c). is a costly process, the addition of MD to the GOS solutions Commercial GOS are generally produced by used as dehydration medium appears as an adequate strategy transgalactosylation of lactose in reactions catalyzed by fungal to increase the T of the powders obtained after spray-drying. and bacterial β-galactosidases (Gänzle et al., 2008). As a result of For this reason, the goal of this work was to use commercial transgalactosylation, GOS of different degrees of polymerization GOS in combination with MD to stabilize Lactobacillus are the main products of reaction, and glucose and lactose plantarum CIDCA 83114 during spray-drying and storage. The remain as secondary products (about 20–25% of the obtained thermophysical properties of GOS:MD matrices were analyzed products) (Tymczyszyn et al., 2011). The presence of these latter by determining water sorption isotherms at room temperature, compounds in the reaction medium leads to a decrease of the T s by differential scanning calorimetry (DSC), and molecular glass transition temperatures (T ) of the obtained GOS. Even g mobility by proton nuclear magnetic resonance ( H-NMR). when commercial GOS are efficient lyoprotectants of lactic acid The investigated matrices were then used as dehydration bacteria during freeze-drying, they fail at stabilizing them during media during spray-drying of L. plantarum CIDCA 83114, storage because of their relatively low T (Tymczyszyn et al., a potentially probiotic strain (Hugo et al., 2008; Golowczyc 2012). Therefore, removing glucose and lactose can contribute et al., 2011a; Kakisu et al., 2013a,b). The obtained powders to increase the T of commercial GOS. Different technologies, were equilibrated at different relative humidities (RH) and including chromatographic operations, selective fermentation, stored at different temperatures. The use of MD to increase or nanofiltration, have been proposed as purification methods the Tg of the dehydration matrices allowed for the first (Cheng et al., 2006; Goulas et al., 2007; Li et al., 2008; Feng time, the spray-drying of a potentially probiotic strain in the et al., 2009; Sanz-Valero, 2009; Botelho-Cunha et al., 2010; presence of GOS. Gosling et al., 2010). However, they are expensive for a large scale production, and in practice, are impracticable for small and MATERIALS AND METHODS medium enterprises. Considering the economical importance of GOS, other strategies should be developed to increase their Galacto-Oligosaccharides and T and thus, improve their capacity to stabilize microorganisms Maltodextrin during storage. R A commercial syrup Cup Oligo H-70 (Kowa Company, Spray-drying is a cost-effective technique that leads to the Tokyo, Japan) kindly donated by Kochi S.A. (Santiago, Chile) transformation of liquid systems (i.e., solutions, dispersions, was used in this study. It contains 75% GOS (d.b.) of emulsions) into dry particulate powders when they get in different degrees of polymerization (DP): 4% high-molecular- contact with a drying medium (air) at high temperatures. weight oligosaccharides (DP ≥ 5); 21% tetrasaccharides (DP4); This technique has been successfully scaled-up, leading to the 47% trisaccharides (DP 3); 23% disaccharides (DP2) including production of flowable powders and reducing the storage and lactose; and 5% monosacharides, including glucose and galactose transportation costs (Santivarangkna et al., 2008). It is commonly (Tymczyszyn et al., 2011). Food grade maltodextrin (MD) used to ensure the stability of food products and has been (dextrose equivalent: 12) was used (Maltodextrin DE 12, increasingly used to stabilize lactic acid bacteria (Golowczyc Ingredion, Buenos Aires, Argentina). et al., 2010, 2011a,b). Using carbohydrates in the dehydration medium contributes to increase the stability of the spray-dried Microorganisms products and bacteria in terms of water activity, moisture L. plantarum CIDCA 83114 was maintained frozen at −80 C content, pH, solubility, hygroscopicity, nutritional composition, in 120 g/L non-fat milk solids. Microbial cells were reactivated glass transition temperature, color, and fluidity (Chen and in MRS broth (de Man et al., 1960) at 37 C before conducting the experiments. For dehydration experiments, reactivated Prebiotics are defined as “non-digestible food ingredients that beneficially affect microorganisms were grown in MRS at 37 C for 24 h (early the host by selectively stimulating the growth and/or activity of one or a limited stationary phase). Then, microorganisms were harvested by number of bacteria in the colon, thus improving the host health” (Gibson and Roberfroid, 1995). centrifugation at 7000 × g at 4 C for 10 min, washed twice with Frontiers in Microbiology | www.frontiersin.org 2 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices 0.85% w/v NaCl (Merck Química, Buenos Aires, Argentina) and [protons signal intensity (I) vs. experimental time (t)] were fitted suspended in the same volume of a solution containing 20% w/w to mono-exponential behavior using Equation 1: GOS and 20% w/w MD. I = A exp (−t/T ) (1) Spray-Drying Procedure GOS:MD solutions containing microorganisms were spray-dried where T is the relaxation time of protons in the polymeric chains in a laboratory-scale spray-dryer (model B290 Büchi mini spray- of the sample and of tightly bound water, and A is a constant. ◦ ◦ dryer) at a constant air inlet temperature of 180 C and an Since no 180 refocus pulse was used in the experiments, the outlet temperature of 75–80 C. Atomization was created by spin-spin relaxation time constants are apparent relaxation time constants, i.e., T . For solid samples, like those used in this work, compressed air at a pressure of 0.5–2 bar and an air flux of 600 L/h. Nozzle diameter was 0.7 mm. Controls: microorganisms it can be assumed that intrinsic T is very close to T (Fullerton and Cameron, 1988). Therefore, T was used for convenience. harvested in the stationary phase, washed with 0.85% w/v NaCl (Merck Química, Buenos Aires, Argentina), suspended in 40% Bacterial Plate Counts MD solutions and spray-dried in the same conditions as the Bacterial cultivability was determined before and after spray- samples. drying and during storage of the equilibrated samples at different temperatures (see below). One gram of spray-dried powder was Humidification Procedure rehydrated in 9 mL of 0.85% w/v NaCl, homogenized for 1 min Opened glass vials containing approximately 1 g of spray-dried in a vortex mixer and maintained at room temperature for samples were equilibrated in sealed jars for 15 days at 20 C in 30 min. Bacterial suspensions were serially diluted and plated atmospheres of the following saturated salts: LiCl, KCH COO, on MRS agar plates. Bacterial counts were determined after 48 h MgCl K CO , and Mg(NO ) (Sigma-Aldrich, Buenos Aires, 2, 2 3 3 2 incubation at 37 C and referred to bacterial counts immediately Argentina), giving RHs of 11, 22, 33, 44, and 52%, respectively. after spray-drying (N ). After having attained the equilibrium, the vials were hermetically Bacterial inactivation rate was determined according to closed to be used in the activities explained below. Equation 2: Glass Transition Temperatures (T ) Log N/N = − kt (2) Glass transition temperatures of the spray-dried samples were determined by DSC (onset values, heating rate: 10 C/min) where N is the CFU/g powder at a given time of storage, N is using a DSC 822 Mettler Toledo calorimeter (Schwerzenbach, the CFU/g powder immediately after spray drying, t is the time Switzerland), calibrated with indium, lead and zinc. Hermetically of storage expressed in days, and k is the bacterial inactivation sealed 40 μL medium pressure pans were used (an empty pan −1 constant expressed in days . was used as reference). Heating and cooling cycles were carried ◦ ◦ out within the −80 to 90 C range at 10 C /min. Thermograms Storage were evaluated using Mettler Star program. An average value of Equilibrated samples at 11, 22, 33, and 44% RH were sealed and ◦ ◦ at least two replicates was reported. The standard deviation for stored at 5 and 20 C for 12 weeks, or at 30 C for 6 weeks. The the glass transition temperature measurement was ±1 C. recovery of cells after different times of storage was analyzed by plate counts. Water Content Determination The bacterial inactivation constant obtained from Equation Karl Fischer titration was carried out at 25 ± 1 C with (2) (k) was then correlated with the inverse of the storage a Karl Fischer titrator DL 31 from Mettler-Toledo (Zurich, temperature (1/T) and fitted to the Arrhenius equation according Switzerland), applying the one-component technique with to Equation 3: Hydranal Titrant Composite 5 from Riedel-de Haën (Seelze, −Ea / RT k = A e (3) Germany). A 95 (1:1) methanol:formamide mixture, obtained from Merck (Darmstadt, Germany), was used as solvent. Sample The linearization of Equation (3) leads to Equation (4): sizes were approximately 100 mg. Ea Ln k = − + ln A (4) Molecular Mobility RT A Bruker mq20 Minispec pulsed H-NMR instrument (Bruker Biospin GmbH, Rheinstetten, Germany), with a 0.47 T magnetic where k is the bacterial inactivation rate constant, A is a frequency −1 field operating at resonance frequency of 20 MHz, was used factor in units of time , Ea is the apparent energy of activation in for measurements. Equilibrated samples were removed from the kJ/mol, R is the gas constant (8.314 J/K mol), and T is the storage desiccators, placed into 10 mm diameter glass tubes and returned temperature in K (Muller et al., 2013). to the desiccators for 24 h prior to analysis. The spin-spin relaxation time (T ) associated to the fast Statistical Analysis relaxing protons (related to the solid matrix and to water tightly All experiments were carried out on duplicate samples using interacting with solids) was measured using a free induction three independent cultures of bacteria. Modeling of Arrhenius decay analysis (FID) after a single 90 pulse. The decay envelopes equation was carried out by two steps linear least squares fit Frontiers in Microbiology | www.frontiersin.org 3 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices (Cohen and Saguy, 1985) and the linear regressions and the goodness of fit (R , residuals and t-test) were carried out using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, 2007). The regression was considered statistically significant if P < 0.1. RESULTS Figure 1A depicts the T of GOS:MD matrices spray-dried in the absence of bacteria and equilibrated at RHs ranging from 11 to 52%. For comparison purposes, information previously obtained for GOS dried without MD was added in the plot (Tymczyszyn et al., 2012). The addition of MD led to a dramatic increase of the T of GOS at all the RHs assayed (Figure 1A). In this regard, at 11% RH the T values of GOS:MD were 30 C higher than those of GOS, and at 52% RH an increase of 20 C with regard to GOS was observed (Figure 1A). Figure 1B depicts the water content of equilibrated spray-dried GOS-MD matrices as a function of RH. Previously obtained results for dehydrated GOS in the absence of MD were also displayed for comparison. It is interesting to note that for RHs greater than 11%, the water content of GOS:MD matrices is noticeably higher than that of GOS matrix. It is also important to point out that the addition of FIGURE 1 | Onset glass transition temperatures (T ) (A) and water MD to commercial GOS allowed the retention of the glassy state ◦ content (in d.b.) (B) of the spray-dried GOS:MD () and freeze-dried up to 44% RH at 30 C (Figure 1A). In contrast, when no MD GOS (♦) matrices (without microorganisms) after equilibration at was added, GOS remained in a glassy state only up to 33% RH at different RHs (30 C). Data corresponding to freeze-dried GOS (♦) were 30 C (Tymczyszyn et al., 2012). These results support the use of obtained previously in the same conditions (Tymczyszyn et al., 2012), and included in the plot for comparison. The dashed line in (A) indicates the GOS:MD formulations as dehydration matrices during bacteria maximum storage temperature, 30 C. spray-drying. T showed a linear relation with the T-T parameter for 2 g GOS:MD matrices equilibrated at 11, 22, 33, and 44% RH, at 5, TABLE 1 | Recovery of L. plantarum CIDCA 83114 after spray-drying in 20, and 30 C (Figure 2). It is interesting to note that samples GOS:MD and MD solutions. equilibrated at 52% RH are out of the linear regression (see ellipse Matrix Before spray-drying After spray-drying Bacterial recovery in Figure 2). In fact, for these samples a noticeable increase of T (CFU/g) (CFU/g) % was observed at all the temperatures assayed (5, 20, and 30 C). The rubbery state of all the samples at 52% RH could explain this 11 10 11 9 GOS-MD 1.2 10 ± 0.5 10 1.12 10 ± 3.5 10 93 behavior. 11 10 10 9 MD (control) 1.2 10 ± 0.5 10 7.75 10 ± 3.5 10 64 The investigated GOS:MD matrices were then used as dehydration media during spray-drying of L. plantarum CIDCA 83114, leading to 93% of bacterial recovery after the thermal high values, particularly for the samples stored at the highest treatment (Table 1). A much lower recovery (64%) was observed temperatures. The k values increase was especially evident for when only MD was included in the dehydration medium. samples stored at 30 C (triangles in Figure 4). This result Figure 3 shows the evolution of Log N/N for L. plantarum indicates that the storage temperature was more relevant than T 0 g CIDCA 83114 spray-dried and equilibrated at 11, 22, 33, and on the inactivation constants. 44 % RH, and stored at 5, 20, and 30 C for 12 weeks (N Figure 5 shows a linear correlation between k (obtained from 11 9 = 1.12 10 ± 3.5 10 CFU/g). As expected, microorganisms the slopes of Figure 3) and the absolute temperature of storage showed the best performance when stored at 5 C (Figure 3A). for each RH. Fitting the Arrhenius equation (Equation 3) allowed In turn, 11% RH was the best condition when stored at 20 and determining the bacterial inactivation constants (k). In turn, with ◦ ◦ 30 C (Figures 3B,C). Storage at 30 C and 44% RH was the most this parameter it is possible to predict the decrease of viability detrimental condition (Figure 3C). (log N/N ) in a given sample at a given water activity, time, In a further step, the slopes of the linear regressions provided and temperature of storage (Equation 2). The linearization of information about the rate of microbial inactivation for each this plot (4) showed that higher slopes were observed with the storage condition (k values) were correlated with T-T parameter increase in RH (Figure 5). This indicates that the higher the (Figure 4). A non-linear increase of k values was observed as storage temperature, the higher their sensitivity to RH. function of T-T parameter. Although most of the samples were From the slopes of the linear regressions obtained in Figure 5, in the glassy state, the inactivation constants showed relatively it was possible to calculate the Ea for each storage condition. Frontiers in Microbiology | www.frontiersin.org 4 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices FIGURE 2 | T for spray-dried GOS:MD matrices as a function of T-T . T were obtained from Figure 1 and T corresponds to the three different ◦ ◦ ◦ storage temperatures: 5 C ( ); 20 C (); and 30 C (△). The points within the ellipse indicate samples equilibrated at 52% RH. These Ea also showed a linear relation with RH (Figure 6), and this information can be useful to calculate the shelf-life of spray- dried samples stored at different temperatures and RH (Muller et al., 2013). DISCUSSION The T s of the commercial GOS used in this work were relatively close to the storage temperatures at all the RH essayed (Figure 1A). The addition of MD to the GOS matrices in a 1:1 ratio appeared as an adequate strategy to increase the T of commercial GOS, leading to the stabilization of L. plantarum CIDCA 83114 during spray-drying (Table 1). Very recently, Rajam and Anandharamakrishnan (2015) adopted a similar strategy to stabilize a strain of L. plantarum during spray-drying. In the mentioned article, the addition of whey proteins and hydrolyzed whey proteins to fructo-oligosaccharides matrices led to a decrease of stickiness and moisture content of the dehydrated samples (Rajam and Anandharamakrishnan, 2015). MD are high molecular weight polysaccharides, produced FIGURE 3 | Relative survival fraction (log N/N ) of L. plantarum by starch hydrolysis. They have different molecular sizes and 0 ◦ ◦ ◦ spray-dried in a GOS:MD matrix and stored at: 5 C (A); 20 C (B); 30 C are classified as a function of their dextrose equivalent units. (C). For each storage temperature, samples were equilibrated at 11 (l), 22 Because of their low viscosity, high solid content, adequate (), 33 (s) and 44 (t)% RH. N = CFU of humidified samples after storage; N water solubility, and high T , they are, in principle, good g = CFU after spray-drying. Solid line indicates the linear regression for each condition. The numbers near the lines indicate the R values. matrices to stabilize samples during spray-drying (Hogan et al., 2001; Sosa et al., 2011). However, using MD matrices without GOS resulted in a noticeable decrease of bacterial cultivability (Table 1). This indicates that the GOS:MD matrices had a double It is interesting to note that although the addition of MD effect. From one side, MD led to an increase of T s (Figure 1A) led to a noticeable increase of T (Figure 1A), the molecular g g and from the other side, the presence of GOS in the matrices mobility of GOS:MD was slightly higher than that reported for led to L. plantarum CIDCA 83114 stabilization (Table 1 and dehydrated GOS (Tymczyszyn et al., 2012). This observation can Figure 3). Although other sugars, including trehalose, glucose, be explained considering the higher hygroscopicity of GOS:MD and inulin, have been previously used to stabilize lactobacilli matrices after equilibration at different RHs (Figure 1B). strains during spray-drying (Sunny-Roberts and Knorr, 2009; Furthermore, the addition of MD to commercial GOS allowed the Golowczyc et al., 2011b; Ying et al., 2012), up to our knowledge, obtaining of glassy matrices at 30 C and 44% RH (Figure 1A). the thermoprotectant effect of GOS has been reported for the first In addition, samples storage led to an increase of the molecular time in this work. mobility (Figure 2). This increased molecular mobility might be Frontiers in Microbiology | www.frontiersin.org 5 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices FIGURE 6 | Activation energy (Ea) as a function of RH. The number near FIGURE 4 | Rate constant of microbial inactivation (k) as a function of the line indicates R . T-T . k was obtained from the slopes of the linear regressions plotted in ◦ ◦ ◦ Figure 3. Microorganisms stored at 5 C ( ); 20 C () and 30 C (△) were included in the plot. that are generally kept at room temperature (i.e., snacks, infant formulas, among others). According to the European Food Safety Agency (EFSA), these values were reported as the minimum bacterial concentration that must be present in the product at the moment of being consumed (Aquilina et al., 2013; Phuapaiboon et al., 2013; Hill et al., 2014; Tripathi and Giri, 2014). In this context, k values represent an important tool to determine the shelf-life of spray- dried L. plantarum CIDCA 83114. The correlation between k and T-T parameters (Figure 4) depicted a similar behavior as that observed previously for Lactobacillus bulgaricus freeze-dried in GOS matrices (Miao et al., 2008; Tymczyszyn et al., 2012). However, the increase of k observed in this work was dependent on the storage temperature FIGURE 5 | Arrhenius plot Ln k vs. 1/T for each storage condition. (Figure 4). L. paracasei freeze-dried in lactose matrices depicted Temperatures are expressed in K. 11% (l); 22% (); 33% (s); 44% (t) RH. a similar behavior, the storage temperature per se having a strong Solid lines indicate the linear regressions for each condition. The numbers 2 influence on the stability of dehydrated samples (Higl et al., close to the lines indicate the R values. All fittings were statistically significant (P < 0.1). 2007). When analyzed as a function of T-T parameter, the molecular mobility of the GOS:MD matrices (Figure 2) and k values responsible for the collapse of GOS:MD, thus indicating that of spray-dried L. plantarum CIDCA 83114 (Figure 4) showed these conditions are not appropriate to preserve microorganisms different patterns. This indicates that the loss of viability at viability during storage. higher storage temperatures could not be explained only by The k parameter obtained from the slopes of the lines in the increase of molecular mobility, pointing out that other Figure 3 represents an important tool directly related with the inactivation mechanisms are certainly present. In this regard, the product shelf-life at storage conditions (temperature and RH). reported higher penetration of oxygen in samples with higher This parameter has been previously determined for Lactobacillus oxidative effects appears as a plausible co-existent mechanism delbrueckii subsp. bulgarius freeze-dried in GOS matrices (Teixeira et al., 1996; Higl et al., 2007; Ying et al., 2011). (Tymczyszyn et al., 2012), L. plantarum and L. delbrueckii According to Figure 5, the effect of temperature on k is subsp bulgaricus immobilized in carboximethylcellullose films conditioned by the water activity. For example among samples containing fructo-oligosaccharides (Romano et al., 2014), and equilibrated at 11% RH, the increase of k at different storage for Lactobacillus paracasei freeze-dried in lactose matrices (Higl temperatures is lower than among samples with a higher water et al., 2007). The results obtained in this work clearly showed content (44% RH) (Figure 5). These results are consistent that storage at low temperatures and low RHs were the best with those reported by Higl et al. (2007). They proposed that conditions (lower k values) to stabilize L. plantarum CIDCA inactivation rates of low water activity samples are independent 83114 (Figure 3). These conditions warrants that at least 6–7 log on the temperatures. CFU of viable microorganisms per gram of product are present The linearization of the Arrhenius equation allowed after 90 days of storage (Figure 3A). This concentration was also determining the Ea for each storage condition from the slopes warranted for microorganisms stored at 20 C at 11 and 22% of the lines (Figure 5). In turn, the linear correlation between RH up to 90 days, and stored at 30 C 11% RH up to 60 days Ea and RH (Figure 6) allowed determining the stability of (Figure 3). This supports their use in the formulation of products dehydrated samples exposed to different storage conditions Frontiers in Microbiology | www.frontiersin.org 6 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices during transport and commercialization. Although this equation 2008; Golowczyc et al., 2011a; Kakisu et al., 2013a,b) and the has been widely used to predict the stability of dehydrated foods, high concentrations of GOS present in the matrices, the obtained it has been scarcely used to predict lactic acid bacteria stability microorganisms may be potentially considered as “synbiotic” at different storage conditions, and thus, it can be considered as products. Taking into account that prebiotics are not hydrolyzed an important contribution of this work (Ying et al., 2012; Muller in the upper part of the gastro-intestinal tract (definition of et al., 2013). prebiotics, Gibson and Roberfroid, 1995), the consumption of potentially probiotic strains embedded in prebiotic matrices appears as an adequate strategy to protect microorganisms when CONCLUSIONS exposed to the harmful conditions of the gastro-intestinal tract. The obtained results strongly support the use of GOS:MD AUTHOR CONTRIBUTIONS matrices as thermoprotectants of L. plantarum CIDCA 83114. Their thermophysical properties have been determined from an NS and CS did the experimental work regarding water content integrative perspective, thus supporting the determination of the determination, molecular mobility, and Tg determination. EG most appropriate storage conditions of dehydrated samples. In and MG did the experimental work and analyzed results obtained spite of that, having appropriate thermophysical properties is a by spray-drying. ET and AGZ coordinated the work (analysis of necessary but not sufficient condition for microbial stabilization. results, discussion, and writing of the manuscript). All authors Bacterial recovery after drying processes is determined by the have approved the final version of the manuscript. presence of protective molecules such as GOS, their stability being favored at high T-T values. At high RH or high storage temperatures (low T-T values), complex deteriorative reactions g ACKNOWLEDGMENTS take place and lead to the increase of inactivation rates. This work was supported by the Argentinean Agency for the From a microbiological point of view, the results obtained Scientific and Technological Promotion (ANPCyT) (Projects support the production of L. plantarum CIDCA 83114, PICT/2014/0912, PICT/2013/1258, PICT/2014/1395), the a potentially probiotic strain, at low cost, using GOS as Argentinean National Research Council (CONICET) (PIP2012- thermoprotectants and spray-drying as dehydration method, with potential applications as functional food ingredient. 2014114-201101-00024 and PIP 112 2015 0100636 CO). NS, EG, MG, CS, AGZ, and ET are members of the research career Considering the inhibitory properties of the studied strain against Escherichia coli O157:H7, Shigella and Salmonella (Hugo et al., CONICET. REFERENCES de Man, J. C., Rogosa, M., and Sharpe, M. E. (1960). 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Edible methylcellulose-based films containing Tymczyszyn. This is an open-access article distributed under the terms of the Creative FOS as vehicles for lactic acid bacteria. Food Res. Int. 64, 560–566. doi: Commons Attribution License (CC BY). The use, distribution or reproduction in 10.1016/j.foodres.2014.07.018 other forums is permitted, provided the original author(s) or licensor are credited Santivarangkna, C., Kulozik, U., and Foerst, P. (2008). Inactivation mechanisms and that the original publication in this journal is cited, in accordance with accepted of lactic acid starter cultures preserved by drying processes. J. Appl. Microbiol. academic practice. No use, distribution or reproduction is permitted which does not 105, 1–13. doi: 10.1111/j.1365-2672.2008.03744.x comply with these terms. Frontiers in Microbiology | www.frontiersin.org 8 May 2016 | Volume 7 | Article 584 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Microbiology Pubmed Central

Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying

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Abstract

ORIGINAL RESEARCH published: 03 May 2016 doi: 10.3389/fmicb.2016.00584 Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying 1 2 2 3 Natalia Sosa , Esteban Gerbino , Marina A. Golowczyc , Carolina Schebor , 2 4 Andrea Gómez-Zavaglia * and E. Elizabeth Tymczyszyn 1 2 Facultad de Bromatología, Universidad Nacional de Entre Ríos, Gualeguaychú, Argentina, Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA, CCT-CONICET), La Plata, Argentina, Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina, Laboratorio de Microbiología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Argentina In this work maltodextrins were added to commercial galacto-oligosaccharides (GOS) in a Edited by: Michael Gänzle, 1:1 ratio and their thermophysical characteristics were analyzed. GOS:MD solutions were University of Alberta, Canada then used as matrices during spray-drying of Lactobacillus plantarum CIDCA 83114. The Reviewed by: obtained powders were equilibrated at different relative humidities (RH) and stored at 5 Fernanda Mozzi, ◦ ◦ Centro de Referencia para and 20 C for 12 weeks, or at 30 C for 6 weeks. The T s of GOS:MD matrices were about Lactobacilos (CERELA-CONICET), 20–30 C higher than those of GOS at RH within 11 and 52%. A linear relation between Argentina the spin-spin relaxation time (T ) and T-T parameter was observed for GOS:MD matrices Sergio I. Martinez-Monteagudo, g South Dakota State University, USA equilibrated at 11, 22, 33, and 44% RH at 5, 20, and 30 C. Spray-drying of L. plantarum *Correspondence: CIDCA 83114 in GOS:MD matrices allowed the recovery of 93% microorganisms. In Andrea Gómez-Zavaglia contrast, only 64% microorganisms were recovered when no GOS were included in the angoza@qui.uc.pt dehydration medium. Survival of L. plantarum CIDCA 83114 during storage showed Specialty section: the best performance for bacteria stored at 5 C. In a further step, the slopes of the This article was submitted to linear regressions provided information about the rate of microbial inactivation for each Food Microbiology, a section of the journal storage condition (k values). This information can be useful to calculate the shelf-life of Frontiers in Microbiology spray-dried starters stored at different temperatures and RH. Using GOS:MD matrices Received: 08 February 2016 as a dehydration medium enhanced the recovery of L. plantarum CIDCA 83114 after Accepted: 11 April 2016 spray-drying. This strategy allowed for the first time the spray-drying stabilization of a Published: 03 May 2016 potentially probiotic strain in the presence of GOS. Citation: Sosa N, Gerbino E, Golowczyc MA, Keywords: galacto-oligosaccharides, maltodextrin, glass transition temperature, spray-drying, lactic acid bacteria Schebor C, Gómez-Zavaglia A and Tymczyszyn EE (2016) Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Abbreviations: GOS, galacto-oligosaccharides; T , glass transition temperature; MD, maltodextrin; DSC, differential Spray-Drying. Front. Microbiol. 7:584. scanning calorimetry; H-NMR, proton nuclear magnetic resonance; RH, relative humidity; d.b., dry basis; DP, degree of doi: 10.3389/fmicb.2016.00584 polymerization; FID, free induction decay analysis; CFU, cell forming units. Frontiers in Microbiology | www.frontiersin.org 1 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices INTRODUCTION Patel, 2008; Fazaeli et al., 2012). However, the thermophysical properties of some commercial GOS are not the most appropriate Galacto-oligosaccharides (GOS) are non-digestible ones to support their use as dehydration media. Their low T oligosaccharides composed of a variable number of galactose (Tymczyszyn et al., 2012) leads to obtaining sticky products units (usually from 2 to 10) and a terminal glucose unit, linked that agglomerate and adhere to the internal wall of the drying mostly by β1-4 and β1-6 bonds (Casci and Rastall, 2006; Vera chamber, thus resulting in a low product yield and low bacterial et al., 2011). They are widely known because of their prebiotic cultivability (Hennigs et al., 2001; Bhandari and Howes, 2005). properties (Gibson and Roberfroid, 1995) and more recently, To overcome this problem, high molecular weight saccharides they demonstrated to enhance the recovery of lactic acid bacteria of high T , including maltodextrins (MD) or starch, have been during dehydration processes (Tymczyszyn et al., 2011, 2012; generally used to avoid the undesirable stickiness of low T sugar Santos et al., 2014a,b,c). The protective properties of GOS solutions (Bhandari et al., 1997; Fazaeli et al., 2012; Rajam and have been explained on the basis of their ability to form glassy Anandharamakrishnan, 2015). matrices of high viscosity and low molecular mobility where Taking into account that commercial GOS generally include molecular interactions are restricted (Tymczyszyn et al., 2011, different amounts of glucose and lactose, and their purification 2012; Golowczyc et al., 2013; Santos et al., 2014a,b,c). is a costly process, the addition of MD to the GOS solutions Commercial GOS are generally produced by used as dehydration medium appears as an adequate strategy transgalactosylation of lactose in reactions catalyzed by fungal to increase the T of the powders obtained after spray-drying. and bacterial β-galactosidases (Gänzle et al., 2008). As a result of For this reason, the goal of this work was to use commercial transgalactosylation, GOS of different degrees of polymerization GOS in combination with MD to stabilize Lactobacillus are the main products of reaction, and glucose and lactose plantarum CIDCA 83114 during spray-drying and storage. The remain as secondary products (about 20–25% of the obtained thermophysical properties of GOS:MD matrices were analyzed products) (Tymczyszyn et al., 2011). The presence of these latter by determining water sorption isotherms at room temperature, compounds in the reaction medium leads to a decrease of the T s by differential scanning calorimetry (DSC), and molecular glass transition temperatures (T ) of the obtained GOS. Even g mobility by proton nuclear magnetic resonance ( H-NMR). when commercial GOS are efficient lyoprotectants of lactic acid The investigated matrices were then used as dehydration bacteria during freeze-drying, they fail at stabilizing them during media during spray-drying of L. plantarum CIDCA 83114, storage because of their relatively low T (Tymczyszyn et al., a potentially probiotic strain (Hugo et al., 2008; Golowczyc 2012). Therefore, removing glucose and lactose can contribute et al., 2011a; Kakisu et al., 2013a,b). The obtained powders to increase the T of commercial GOS. Different technologies, were equilibrated at different relative humidities (RH) and including chromatographic operations, selective fermentation, stored at different temperatures. The use of MD to increase or nanofiltration, have been proposed as purification methods the Tg of the dehydration matrices allowed for the first (Cheng et al., 2006; Goulas et al., 2007; Li et al., 2008; Feng time, the spray-drying of a potentially probiotic strain in the et al., 2009; Sanz-Valero, 2009; Botelho-Cunha et al., 2010; presence of GOS. Gosling et al., 2010). However, they are expensive for a large scale production, and in practice, are impracticable for small and MATERIALS AND METHODS medium enterprises. Considering the economical importance of GOS, other strategies should be developed to increase their Galacto-Oligosaccharides and T and thus, improve their capacity to stabilize microorganisms Maltodextrin during storage. R A commercial syrup Cup Oligo H-70 (Kowa Company, Spray-drying is a cost-effective technique that leads to the Tokyo, Japan) kindly donated by Kochi S.A. (Santiago, Chile) transformation of liquid systems (i.e., solutions, dispersions, was used in this study. It contains 75% GOS (d.b.) of emulsions) into dry particulate powders when they get in different degrees of polymerization (DP): 4% high-molecular- contact with a drying medium (air) at high temperatures. weight oligosaccharides (DP ≥ 5); 21% tetrasaccharides (DP4); This technique has been successfully scaled-up, leading to the 47% trisaccharides (DP 3); 23% disaccharides (DP2) including production of flowable powders and reducing the storage and lactose; and 5% monosacharides, including glucose and galactose transportation costs (Santivarangkna et al., 2008). It is commonly (Tymczyszyn et al., 2011). Food grade maltodextrin (MD) used to ensure the stability of food products and has been (dextrose equivalent: 12) was used (Maltodextrin DE 12, increasingly used to stabilize lactic acid bacteria (Golowczyc Ingredion, Buenos Aires, Argentina). et al., 2010, 2011a,b). Using carbohydrates in the dehydration medium contributes to increase the stability of the spray-dried Microorganisms products and bacteria in terms of water activity, moisture L. plantarum CIDCA 83114 was maintained frozen at −80 C content, pH, solubility, hygroscopicity, nutritional composition, in 120 g/L non-fat milk solids. Microbial cells were reactivated glass transition temperature, color, and fluidity (Chen and in MRS broth (de Man et al., 1960) at 37 C before conducting the experiments. For dehydration experiments, reactivated Prebiotics are defined as “non-digestible food ingredients that beneficially affect microorganisms were grown in MRS at 37 C for 24 h (early the host by selectively stimulating the growth and/or activity of one or a limited stationary phase). Then, microorganisms were harvested by number of bacteria in the colon, thus improving the host health” (Gibson and Roberfroid, 1995). centrifugation at 7000 × g at 4 C for 10 min, washed twice with Frontiers in Microbiology | www.frontiersin.org 2 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices 0.85% w/v NaCl (Merck Química, Buenos Aires, Argentina) and [protons signal intensity (I) vs. experimental time (t)] were fitted suspended in the same volume of a solution containing 20% w/w to mono-exponential behavior using Equation 1: GOS and 20% w/w MD. I = A exp (−t/T ) (1) Spray-Drying Procedure GOS:MD solutions containing microorganisms were spray-dried where T is the relaxation time of protons in the polymeric chains in a laboratory-scale spray-dryer (model B290 Büchi mini spray- of the sample and of tightly bound water, and A is a constant. ◦ ◦ dryer) at a constant air inlet temperature of 180 C and an Since no 180 refocus pulse was used in the experiments, the outlet temperature of 75–80 C. Atomization was created by spin-spin relaxation time constants are apparent relaxation time constants, i.e., T . For solid samples, like those used in this work, compressed air at a pressure of 0.5–2 bar and an air flux of 600 L/h. Nozzle diameter was 0.7 mm. Controls: microorganisms it can be assumed that intrinsic T is very close to T (Fullerton and Cameron, 1988). Therefore, T was used for convenience. harvested in the stationary phase, washed with 0.85% w/v NaCl (Merck Química, Buenos Aires, Argentina), suspended in 40% Bacterial Plate Counts MD solutions and spray-dried in the same conditions as the Bacterial cultivability was determined before and after spray- samples. drying and during storage of the equilibrated samples at different temperatures (see below). One gram of spray-dried powder was Humidification Procedure rehydrated in 9 mL of 0.85% w/v NaCl, homogenized for 1 min Opened glass vials containing approximately 1 g of spray-dried in a vortex mixer and maintained at room temperature for samples were equilibrated in sealed jars for 15 days at 20 C in 30 min. Bacterial suspensions were serially diluted and plated atmospheres of the following saturated salts: LiCl, KCH COO, on MRS agar plates. Bacterial counts were determined after 48 h MgCl K CO , and Mg(NO ) (Sigma-Aldrich, Buenos Aires, 2, 2 3 3 2 incubation at 37 C and referred to bacterial counts immediately Argentina), giving RHs of 11, 22, 33, 44, and 52%, respectively. after spray-drying (N ). After having attained the equilibrium, the vials were hermetically Bacterial inactivation rate was determined according to closed to be used in the activities explained below. Equation 2: Glass Transition Temperatures (T ) Log N/N = − kt (2) Glass transition temperatures of the spray-dried samples were determined by DSC (onset values, heating rate: 10 C/min) where N is the CFU/g powder at a given time of storage, N is using a DSC 822 Mettler Toledo calorimeter (Schwerzenbach, the CFU/g powder immediately after spray drying, t is the time Switzerland), calibrated with indium, lead and zinc. Hermetically of storage expressed in days, and k is the bacterial inactivation sealed 40 μL medium pressure pans were used (an empty pan −1 constant expressed in days . was used as reference). Heating and cooling cycles were carried ◦ ◦ out within the −80 to 90 C range at 10 C /min. Thermograms Storage were evaluated using Mettler Star program. An average value of Equilibrated samples at 11, 22, 33, and 44% RH were sealed and ◦ ◦ at least two replicates was reported. The standard deviation for stored at 5 and 20 C for 12 weeks, or at 30 C for 6 weeks. The the glass transition temperature measurement was ±1 C. recovery of cells after different times of storage was analyzed by plate counts. Water Content Determination The bacterial inactivation constant obtained from Equation Karl Fischer titration was carried out at 25 ± 1 C with (2) (k) was then correlated with the inverse of the storage a Karl Fischer titrator DL 31 from Mettler-Toledo (Zurich, temperature (1/T) and fitted to the Arrhenius equation according Switzerland), applying the one-component technique with to Equation 3: Hydranal Titrant Composite 5 from Riedel-de Haën (Seelze, −Ea / RT k = A e (3) Germany). A 95 (1:1) methanol:formamide mixture, obtained from Merck (Darmstadt, Germany), was used as solvent. Sample The linearization of Equation (3) leads to Equation (4): sizes were approximately 100 mg. Ea Ln k = − + ln A (4) Molecular Mobility RT A Bruker mq20 Minispec pulsed H-NMR instrument (Bruker Biospin GmbH, Rheinstetten, Germany), with a 0.47 T magnetic where k is the bacterial inactivation rate constant, A is a frequency −1 field operating at resonance frequency of 20 MHz, was used factor in units of time , Ea is the apparent energy of activation in for measurements. Equilibrated samples were removed from the kJ/mol, R is the gas constant (8.314 J/K mol), and T is the storage desiccators, placed into 10 mm diameter glass tubes and returned temperature in K (Muller et al., 2013). to the desiccators for 24 h prior to analysis. The spin-spin relaxation time (T ) associated to the fast Statistical Analysis relaxing protons (related to the solid matrix and to water tightly All experiments were carried out on duplicate samples using interacting with solids) was measured using a free induction three independent cultures of bacteria. Modeling of Arrhenius decay analysis (FID) after a single 90 pulse. The decay envelopes equation was carried out by two steps linear least squares fit Frontiers in Microbiology | www.frontiersin.org 3 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices (Cohen and Saguy, 1985) and the linear regressions and the goodness of fit (R , residuals and t-test) were carried out using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, 2007). The regression was considered statistically significant if P < 0.1. RESULTS Figure 1A depicts the T of GOS:MD matrices spray-dried in the absence of bacteria and equilibrated at RHs ranging from 11 to 52%. For comparison purposes, information previously obtained for GOS dried without MD was added in the plot (Tymczyszyn et al., 2012). The addition of MD led to a dramatic increase of the T of GOS at all the RHs assayed (Figure 1A). In this regard, at 11% RH the T values of GOS:MD were 30 C higher than those of GOS, and at 52% RH an increase of 20 C with regard to GOS was observed (Figure 1A). Figure 1B depicts the water content of equilibrated spray-dried GOS-MD matrices as a function of RH. Previously obtained results for dehydrated GOS in the absence of MD were also displayed for comparison. It is interesting to note that for RHs greater than 11%, the water content of GOS:MD matrices is noticeably higher than that of GOS matrix. It is also important to point out that the addition of FIGURE 1 | Onset glass transition temperatures (T ) (A) and water MD to commercial GOS allowed the retention of the glassy state ◦ content (in d.b.) (B) of the spray-dried GOS:MD () and freeze-dried up to 44% RH at 30 C (Figure 1A). In contrast, when no MD GOS (♦) matrices (without microorganisms) after equilibration at was added, GOS remained in a glassy state only up to 33% RH at different RHs (30 C). Data corresponding to freeze-dried GOS (♦) were 30 C (Tymczyszyn et al., 2012). These results support the use of obtained previously in the same conditions (Tymczyszyn et al., 2012), and included in the plot for comparison. The dashed line in (A) indicates the GOS:MD formulations as dehydration matrices during bacteria maximum storage temperature, 30 C. spray-drying. T showed a linear relation with the T-T parameter for 2 g GOS:MD matrices equilibrated at 11, 22, 33, and 44% RH, at 5, TABLE 1 | Recovery of L. plantarum CIDCA 83114 after spray-drying in 20, and 30 C (Figure 2). It is interesting to note that samples GOS:MD and MD solutions. equilibrated at 52% RH are out of the linear regression (see ellipse Matrix Before spray-drying After spray-drying Bacterial recovery in Figure 2). In fact, for these samples a noticeable increase of T (CFU/g) (CFU/g) % was observed at all the temperatures assayed (5, 20, and 30 C). The rubbery state of all the samples at 52% RH could explain this 11 10 11 9 GOS-MD 1.2 10 ± 0.5 10 1.12 10 ± 3.5 10 93 behavior. 11 10 10 9 MD (control) 1.2 10 ± 0.5 10 7.75 10 ± 3.5 10 64 The investigated GOS:MD matrices were then used as dehydration media during spray-drying of L. plantarum CIDCA 83114, leading to 93% of bacterial recovery after the thermal high values, particularly for the samples stored at the highest treatment (Table 1). A much lower recovery (64%) was observed temperatures. The k values increase was especially evident for when only MD was included in the dehydration medium. samples stored at 30 C (triangles in Figure 4). This result Figure 3 shows the evolution of Log N/N for L. plantarum indicates that the storage temperature was more relevant than T 0 g CIDCA 83114 spray-dried and equilibrated at 11, 22, 33, and on the inactivation constants. 44 % RH, and stored at 5, 20, and 30 C for 12 weeks (N Figure 5 shows a linear correlation between k (obtained from 11 9 = 1.12 10 ± 3.5 10 CFU/g). As expected, microorganisms the slopes of Figure 3) and the absolute temperature of storage showed the best performance when stored at 5 C (Figure 3A). for each RH. Fitting the Arrhenius equation (Equation 3) allowed In turn, 11% RH was the best condition when stored at 20 and determining the bacterial inactivation constants (k). In turn, with ◦ ◦ 30 C (Figures 3B,C). Storage at 30 C and 44% RH was the most this parameter it is possible to predict the decrease of viability detrimental condition (Figure 3C). (log N/N ) in a given sample at a given water activity, time, In a further step, the slopes of the linear regressions provided and temperature of storage (Equation 2). The linearization of information about the rate of microbial inactivation for each this plot (4) showed that higher slopes were observed with the storage condition (k values) were correlated with T-T parameter increase in RH (Figure 5). This indicates that the higher the (Figure 4). A non-linear increase of k values was observed as storage temperature, the higher their sensitivity to RH. function of T-T parameter. Although most of the samples were From the slopes of the linear regressions obtained in Figure 5, in the glassy state, the inactivation constants showed relatively it was possible to calculate the Ea for each storage condition. Frontiers in Microbiology | www.frontiersin.org 4 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices FIGURE 2 | T for spray-dried GOS:MD matrices as a function of T-T . T were obtained from Figure 1 and T corresponds to the three different ◦ ◦ ◦ storage temperatures: 5 C ( ); 20 C (); and 30 C (△). The points within the ellipse indicate samples equilibrated at 52% RH. These Ea also showed a linear relation with RH (Figure 6), and this information can be useful to calculate the shelf-life of spray- dried samples stored at different temperatures and RH (Muller et al., 2013). DISCUSSION The T s of the commercial GOS used in this work were relatively close to the storage temperatures at all the RH essayed (Figure 1A). The addition of MD to the GOS matrices in a 1:1 ratio appeared as an adequate strategy to increase the T of commercial GOS, leading to the stabilization of L. plantarum CIDCA 83114 during spray-drying (Table 1). Very recently, Rajam and Anandharamakrishnan (2015) adopted a similar strategy to stabilize a strain of L. plantarum during spray-drying. In the mentioned article, the addition of whey proteins and hydrolyzed whey proteins to fructo-oligosaccharides matrices led to a decrease of stickiness and moisture content of the dehydrated samples (Rajam and Anandharamakrishnan, 2015). MD are high molecular weight polysaccharides, produced FIGURE 3 | Relative survival fraction (log N/N ) of L. plantarum by starch hydrolysis. They have different molecular sizes and 0 ◦ ◦ ◦ spray-dried in a GOS:MD matrix and stored at: 5 C (A); 20 C (B); 30 C are classified as a function of their dextrose equivalent units. (C). For each storage temperature, samples were equilibrated at 11 (l), 22 Because of their low viscosity, high solid content, adequate (), 33 (s) and 44 (t)% RH. N = CFU of humidified samples after storage; N water solubility, and high T , they are, in principle, good g = CFU after spray-drying. Solid line indicates the linear regression for each condition. The numbers near the lines indicate the R values. matrices to stabilize samples during spray-drying (Hogan et al., 2001; Sosa et al., 2011). However, using MD matrices without GOS resulted in a noticeable decrease of bacterial cultivability (Table 1). This indicates that the GOS:MD matrices had a double It is interesting to note that although the addition of MD effect. From one side, MD led to an increase of T s (Figure 1A) led to a noticeable increase of T (Figure 1A), the molecular g g and from the other side, the presence of GOS in the matrices mobility of GOS:MD was slightly higher than that reported for led to L. plantarum CIDCA 83114 stabilization (Table 1 and dehydrated GOS (Tymczyszyn et al., 2012). This observation can Figure 3). Although other sugars, including trehalose, glucose, be explained considering the higher hygroscopicity of GOS:MD and inulin, have been previously used to stabilize lactobacilli matrices after equilibration at different RHs (Figure 1B). strains during spray-drying (Sunny-Roberts and Knorr, 2009; Furthermore, the addition of MD to commercial GOS allowed the Golowczyc et al., 2011b; Ying et al., 2012), up to our knowledge, obtaining of glassy matrices at 30 C and 44% RH (Figure 1A). the thermoprotectant effect of GOS has been reported for the first In addition, samples storage led to an increase of the molecular time in this work. mobility (Figure 2). This increased molecular mobility might be Frontiers in Microbiology | www.frontiersin.org 5 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices FIGURE 6 | Activation energy (Ea) as a function of RH. The number near FIGURE 4 | Rate constant of microbial inactivation (k) as a function of the line indicates R . T-T . k was obtained from the slopes of the linear regressions plotted in ◦ ◦ ◦ Figure 3. Microorganisms stored at 5 C ( ); 20 C () and 30 C (△) were included in the plot. that are generally kept at room temperature (i.e., snacks, infant formulas, among others). According to the European Food Safety Agency (EFSA), these values were reported as the minimum bacterial concentration that must be present in the product at the moment of being consumed (Aquilina et al., 2013; Phuapaiboon et al., 2013; Hill et al., 2014; Tripathi and Giri, 2014). In this context, k values represent an important tool to determine the shelf-life of spray- dried L. plantarum CIDCA 83114. The correlation between k and T-T parameters (Figure 4) depicted a similar behavior as that observed previously for Lactobacillus bulgaricus freeze-dried in GOS matrices (Miao et al., 2008; Tymczyszyn et al., 2012). However, the increase of k observed in this work was dependent on the storage temperature FIGURE 5 | Arrhenius plot Ln k vs. 1/T for each storage condition. (Figure 4). L. paracasei freeze-dried in lactose matrices depicted Temperatures are expressed in K. 11% (l); 22% (); 33% (s); 44% (t) RH. a similar behavior, the storage temperature per se having a strong Solid lines indicate the linear regressions for each condition. The numbers 2 influence on the stability of dehydrated samples (Higl et al., close to the lines indicate the R values. All fittings were statistically significant (P < 0.1). 2007). When analyzed as a function of T-T parameter, the molecular mobility of the GOS:MD matrices (Figure 2) and k values responsible for the collapse of GOS:MD, thus indicating that of spray-dried L. plantarum CIDCA 83114 (Figure 4) showed these conditions are not appropriate to preserve microorganisms different patterns. This indicates that the loss of viability at viability during storage. higher storage temperatures could not be explained only by The k parameter obtained from the slopes of the lines in the increase of molecular mobility, pointing out that other Figure 3 represents an important tool directly related with the inactivation mechanisms are certainly present. In this regard, the product shelf-life at storage conditions (temperature and RH). reported higher penetration of oxygen in samples with higher This parameter has been previously determined for Lactobacillus oxidative effects appears as a plausible co-existent mechanism delbrueckii subsp. bulgarius freeze-dried in GOS matrices (Teixeira et al., 1996; Higl et al., 2007; Ying et al., 2011). (Tymczyszyn et al., 2012), L. plantarum and L. delbrueckii According to Figure 5, the effect of temperature on k is subsp bulgaricus immobilized in carboximethylcellullose films conditioned by the water activity. For example among samples containing fructo-oligosaccharides (Romano et al., 2014), and equilibrated at 11% RH, the increase of k at different storage for Lactobacillus paracasei freeze-dried in lactose matrices (Higl temperatures is lower than among samples with a higher water et al., 2007). The results obtained in this work clearly showed content (44% RH) (Figure 5). These results are consistent that storage at low temperatures and low RHs were the best with those reported by Higl et al. (2007). They proposed that conditions (lower k values) to stabilize L. plantarum CIDCA inactivation rates of low water activity samples are independent 83114 (Figure 3). These conditions warrants that at least 6–7 log on the temperatures. CFU of viable microorganisms per gram of product are present The linearization of the Arrhenius equation allowed after 90 days of storage (Figure 3A). This concentration was also determining the Ea for each storage condition from the slopes warranted for microorganisms stored at 20 C at 11 and 22% of the lines (Figure 5). In turn, the linear correlation between RH up to 90 days, and stored at 30 C 11% RH up to 60 days Ea and RH (Figure 6) allowed determining the stability of (Figure 3). This supports their use in the formulation of products dehydrated samples exposed to different storage conditions Frontiers in Microbiology | www.frontiersin.org 6 May 2016 | Volume 7 | Article 584 Sosa et al. L. plantarum Spray-Dried in GOS: Maltodextrin Matrices during transport and commercialization. Although this equation 2008; Golowczyc et al., 2011a; Kakisu et al., 2013a,b) and the has been widely used to predict the stability of dehydrated foods, high concentrations of GOS present in the matrices, the obtained it has been scarcely used to predict lactic acid bacteria stability microorganisms may be potentially considered as “synbiotic” at different storage conditions, and thus, it can be considered as products. Taking into account that prebiotics are not hydrolyzed an important contribution of this work (Ying et al., 2012; Muller in the upper part of the gastro-intestinal tract (definition of et al., 2013). prebiotics, Gibson and Roberfroid, 1995), the consumption of potentially probiotic strains embedded in prebiotic matrices appears as an adequate strategy to protect microorganisms when CONCLUSIONS exposed to the harmful conditions of the gastro-intestinal tract. The obtained results strongly support the use of GOS:MD AUTHOR CONTRIBUTIONS matrices as thermoprotectants of L. plantarum CIDCA 83114. Their thermophysical properties have been determined from an NS and CS did the experimental work regarding water content integrative perspective, thus supporting the determination of the determination, molecular mobility, and Tg determination. EG most appropriate storage conditions of dehydrated samples. In and MG did the experimental work and analyzed results obtained spite of that, having appropriate thermophysical properties is a by spray-drying. ET and AGZ coordinated the work (analysis of necessary but not sufficient condition for microbial stabilization. results, discussion, and writing of the manuscript). All authors Bacterial recovery after drying processes is determined by the have approved the final version of the manuscript. presence of protective molecules such as GOS, their stability being favored at high T-T values. At high RH or high storage temperatures (low T-T values), complex deteriorative reactions g ACKNOWLEDGMENTS take place and lead to the increase of inactivation rates. This work was supported by the Argentinean Agency for the From a microbiological point of view, the results obtained Scientific and Technological Promotion (ANPCyT) (Projects support the production of L. plantarum CIDCA 83114, PICT/2014/0912, PICT/2013/1258, PICT/2014/1395), the a potentially probiotic strain, at low cost, using GOS as Argentinean National Research Council (CONICET) (PIP2012- thermoprotectants and spray-drying as dehydration method, with potential applications as functional food ingredient. 2014114-201101-00024 and PIP 112 2015 0100636 CO). NS, EG, MG, CS, AGZ, and ET are members of the research career Considering the inhibitory properties of the studied strain against Escherichia coli O157:H7, Shigella and Salmonella (Hugo et al., CONICET. REFERENCES de Man, J. C., Rogosa, M., and Sharpe, M. E. (1960). 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Edible methylcellulose-based films containing Tymczyszyn. This is an open-access article distributed under the terms of the Creative FOS as vehicles for lactic acid bacteria. Food Res. Int. 64, 560–566. doi: Commons Attribution License (CC BY). The use, distribution or reproduction in 10.1016/j.foodres.2014.07.018 other forums is permitted, provided the original author(s) or licensor are credited Santivarangkna, C., Kulozik, U., and Foerst, P. (2008). Inactivation mechanisms and that the original publication in this journal is cited, in accordance with accepted of lactic acid starter cultures preserved by drying processes. J. Appl. Microbiol. academic practice. No use, distribution or reproduction is permitted which does not 105, 1–13. doi: 10.1111/j.1365-2672.2008.03744.x comply with these terms. Frontiers in Microbiology | www.frontiersin.org 8 May 2016 | Volume 7 | Article 584

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Published: May 3, 2016

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