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Influence of Osmotic Stress and Encapsulating Materials on the Stability of Autochthonous Lactobacillus plantarum after Spray Drying
Paola Bustos & Rodrigo Brquez
a a a

Chemical Engineering Department, University of Concepcin, Concepcion, Chile Version of record first published: 10 Jan 2013.

To cite this article: Paola Bustos & Rodrigo Brquez (2013): Influence of Osmotic Stress and Encapsulating Materials on the Stability of Autochthonous Lactobacillus plantarum after Spray Drying, Drying Technology: An International Journal, 31:1, 57-66 To link to this article: http://dx.doi.org/10.1080/07373937.2012.717325

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Drying Technology, 31: 5766, 2013 Copyright # 2013 Taylor & Francis Group, LLC ISSN: 0737-3937 print=1532-2300 online DOI: 10.1080/07373937.2012.717325

Inuence of Osmotic Stress and Encapsulating Materials on the Stability of Autochthonous Lactobacillus plantarum after Spray Drying
rquez Paola Bustos and Rodrigo Bo
n, Concepcion, Chile Chemical Engineering Department, University of Concepcio

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With the aim of producing a functional sh food, autochthonous Lactobacillus plantarum LPS 47, isolated from healthy Chilean salmon, was stabilized using spray drying. In order to increase the resistance of microorganisms to high temperatures, osmotic stress during fermentation and different protective agents were evaluated (lactic solids: skim milk and whey, maltodextrin, pectin, and arabic gum). We found that bacteria that were not exposed to osmotic stress during culture, and that were spray-dried in the presence of cheese whey (20% w/w) as a protective material, gave the best results in terms of viability, humidity, and stability during storage. Dehydrated material reached a viability of approximately 109 CFU/g (colony-forming units per gram) and less than 5% humidity, values that were maintained over 10 weeks of storage at 4 C in a non-controlled atmosphere. Results show that it is possible to obtain a stable dehydrated probiotic product, with high concentration of microorganism, using spray drying and whey as a protective agent, without causing osmotic stress, by harvesting the biomass during the stationary phase. Keywords Encapsulation; Osmotic stress; Probiotics; Spray drying

INTRODUCTION Chilean salmon exports are positioned in second place internationally (after Norway), accounting for 2,200 MUS$ during 2007.[1] Exported salmon are high-quality products, and salmon farms strive to avoid opportunistic diseases caused by pathogens including Aeromonas salmonicida, Vibrio anguillarum, Vibrio salmonicida, and Yersinia ruckeri, using sanitary prophylaxis, disinfection, and chemotherapy. The chemotherapy is largely in the form of antibiotics,[2] whose national use has been estimated to be 550 ton=yr.[3] Because of problems associated with these kind of substances (e.g., antibiotic resistance), there is growing interest in nding a natural way to protect species, ensuring a product free of disease and=or traces of undesirable substances. In recent years, reports have suggested

Correspondence: Paola Bustos, Chemical Engineering Department, University of Concepcion, Concepcion, Chile; E-mail: paobusto@udec.cl

that problems in aquaculture could be remedied by the use of probiotics.[25] As stated by Ringo,[2] probiotic mechanisms include the production of inhibitory substances against pathogens, such as organic acids and bacteriocins, and competition for essential nutrients and enzymes. Kesarcodi-Watson[5] also adds: competition for attachment sites, alteration of the enzymatic activity of pathogens, inmuno-stimulatory functions and nutritional benets, such as improving feed digestibility and feed utilization. Some species of lactic acid bacteria (LAB) and bidobacteria have been widely used as probiotics for human and terrestrial animals and, recently, several works have focused on the use of these as regulators of larval development, growth, and survival during the rst weeks of life in sh,[2,4,5] being incorporated, for example, into a sh feed formulation.[6] Probiotic LAB are marketed in either frozen or dried form, the latter commonly produced by freeze drying. In order to reduce the time and energy costs associated with this process, several authors have recommend spray drying as the most suitable alternative for high production rates, as it costs 80% less than freeze drying.[712] Although spray drying has the principal disadvantage of loss of viability caused by thermal and dehydration inactivation,[13,14] microencapsulation by spray drying greatly reduces the impact on microorganisms and, compared to other microencapsulation techniques, spray drying offers the advantage of producing microcapsules in a continuous and exible process.[11,15,16] In microencapsulation by spray drying, the biomass is homogenized with the carrier material (including carbohydrates, polymers, gums, lipids, or mixtures thereof)[15,17,18] at different ratios. According to Desai and Park, the composition of the coating material is the main determinant of the functional properties of the microcapsule.[11] The mixture is then fed into a spray dryer and atomized with a nozzle or spinning wheel. Water is evaporated and microcapsules are collected after they fall to the bottom of the drier.[11,15,17,19]

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In addition to microencapsulation, the pre-adaptation to non-lethal levels of stress as a tool to reduce mortality during spray drying has been also studied,[2022] and exposure to three main types of stress (acidic, thermal, and saline) has been investigated.[2124] In all forms of stress, the way in which the bacterium protects itself is related to over-expression of specic heat shock proteins and, in some cases, with the production and assimilation of compatible solutes,[25,26] the latter being especially related to saline stress, although it has been noted that accumulation does not take place rapidly, hence it is necessary to promote their production using a pre-drying step.[20,27,28] Thermal stress, which involves the exposure of bacteria to temperatures below those which could cause death, has been used successfully in a variety of probiotic microorganisms where it has been observed that, not only are specic proteins produced as already mentioned, but also there are changes in the cell membrane, mainly related to the saturation and the length of the fatty acids of which it is composed, and which maintain the uidity of the membrane at an optimum level.[29] The operating conditions of the dryer, and also conditions of the feed, seem to inuence the quality of the nal product.[28,29] Process parameters studied include: air-ow pattern (co-current, counter-ow, and mixed ow), nozzle pressure, residence time, feed rate, and in-outlet air temperature.[16,30,31] Of all of these, it has been reported that air temperature is the parameter that most affects the viability of microorganisms,[8,22,28,31] although Telang and Thorat reported that feed ow rate also has an effect on the viability of the product.[16] The objective of this study was to evaluate different encapsulating materials and the effect of different levels of saline stress during growth of autochthonous Lactobacillus plantarum, as methods to improve its resistance during drying and storage in a non-controlled atmosphere. Additionally, process parameters of the spray drier were studied in order to assure a good yield, low humidity, and prevention of excessive damage to bacteria. Finally, behavior of the dehydrated strain was evaluated in terms of growth, acidication, and inhibitory capacity, all desirable characteristics to an organism with probiotic purposes. MATERIALS AND METHODS Organisms and Culture Medium Lactobacillus plantarum LPS 47 isolated from healthy salmon from the south of Chile were grown, in suspension, in whey permeate (from dairy company MULPULMO) supplemented with peptone, yeast extract, and minerals (all from Merck). Different quantities of sodium chloride (NaCl, Merck) were added to the basal medium in order to reach saline stress levels of either 0.25, 0.50, or 0.75 molar (M).

TABLE 1 Composition of protective solutions (values are given in weight percentage) Whey Maltodextrine (W) (M) W20 20 W30 30 W40 40 W50 50 W 20 WM 13 SM WMG 11 WMP 12.8 7 7 6 6.7 Skim milk (S) 13 Arabic gum (G) 3 Pectin (P) 0.5

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Yersinia ruckeri was isolated during outbreaks of Yersiniosis in sh farms in the south of Chile in 2005. Bacteria were freeze-dried and stored at the Laboratory of Ictio n, Chile. pathology at University of Concepcio Culture Conditions and Harvesting Stock cultures of Lactobacillus plantarum were stored at 20 C in a skimmed milk suspension supplemented with 10% yeast extract. Inoculum (0.18% v=v) consisted of a cell suspension of 2412 h-aged pre-culture washed and resuspended in saline buffer (0.9% w=v NaCl) and adjusted to an optical density at 540 nm (OD540) of 0.500 ($105 CFU=ml). Microorganisms were grown in 500 ml of media culture under agitation at 25 C for 30 hours in order for the bacteria to achieve stationary growth phase. Cultures were then harvested by centrifugation at 8,000 rpm for 20 minutes (Heraues, Germany). The supernatant was discarded and the biomass was resuspended in 100 ml of saline buffer. Protective Materials Resuspended bacteria (100 ml) were added to protective solutions (400 ml), which were prepared by adding the protective substances to the saline buffer and gently stirring until dissolved (1015 min). The percentages of different substances are described in Table 1. Whey was supplied by the dairy company MULPULMO (protein 1214%, lipids 0.51.5%, lactose 7074%, minerals 68%, moisture 23%), maltodextrine was obtained from Inducorn A.S (Globe1 019100, 1014% degree of esterication), skimmed milk (Merck), pectin (Sigma Aldrich, LM, 3339% degree of esterication), and arabic gum (Merck). Additionally, two control cultures (osmotically unstressed bacteria), grown at 25 C and 32 C, were prepared in order to study the effect of thermal stress. In both

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cases, at the end of fermentation, the bacterial pellet was resuspended in 500 ml of saline buffer alone and transferred directly to the drier. Spray Drying Samples of 500 ml of protected bacteria were dried in a pilot-scale spray drier of local construction (stainless-steel, evaporative capacity of 2.8 kg=h water, two uid nozzle atomizers with an inside diameter of 0.8 mm). The effects of feed solids content (20, 30, 40, and 50% w=w), pH of feed (pH 4.5, pH 6.0, and pH 7.0), atomizing feed pressure (1, 2, and 3 bar), and outlet air temperature (70, 80, and 90 C) on the operation of the equipment were studied separately and evaluated in terms of yield, humidity, and viability of nal product. All assays were performed using cheese whey as the protective material. Inlet temperature was kept at 180 2 C. Storage Dried samples were packed in sealed plastic asks protected from light at 4 C. Viability was measured over 10 weeks. Analysis Yield To calculate the yield from the experiments, the mass of product was divided by the total mass of solids fed to the spray drier. Results were expressed as percentage. Humidity Moisture content of the powders was measured gravimetrically by oven drying at 135 C until constant weight.[32] Viability Before spray drying, cell viability was measured according to the standard MRS agar plate count method. After drying, a rst dilution was prepared from around 0.5 g of spray-dried bacteria reconstituted in 4 ml of saline solution and after 15 minutes at ambient temperature the following dilutions were made. Plates were incubated for 48 hours at 25 C in aerobic atmosphere. Viability was measured in CFU=g. Cell Damage As membrane cell damage is related to sensitivity to NaCl, to investigate cellular membrane damage resulting from osmotic stress and the spray-drying process, Gardiners method was used.[24] Solutions of resuspended dried bacteria, the same that were used to measure viability, were pour plated onto MRS agar supplemented with 4% NaCl. Plates were counted after three days of incubation at 37 C, colony sizes and numbers were compared to colony sizes and numbers on MRS plates.

Growth after Spray Drying Once the best protective agent had been dened, the growth capacity of the dehydrated strain was evaluated. Around 0.1 g of powder were dissolved in 200 ml of culture medium, gently stirred, and allowed to stand for 15 minutes before the experiments start. Viability and decrease in pH were measured over 48 hours. Results were compared with those obtained from a stock culture strain. Bacterial growth was modeled using the Gompertz model.[33] N ln N0      lm exp1 k t 1 1 A exp exp A 

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In Eq. (1), N are CFU=ml at time t, N0 are CFU=ml at the beginning of culture, lm is maximal growth rate, and k and A are lag time and concentration at stationary phase of growth, respectively. Inhibition of Yersinia ruckeri In order to compare probiotic capacity of dehydrated and fresh strain (as control), the effect on growth of Yersinia ruckeri was evaluated. For this, the microorganism inoculum was adjusted to an OD540 of 0.1000.200 in order to assure an initial concentration of $104 CFU=ml. Lactobacillus and Yersinia strains were both cultured in Tryptic Soy broth (Merck) for 48 hours under the same conditions of culture that have been stated previously. Viable microorganisms were counted on Tryptic soy-agar plates to which 1% of crystal violet was added. Statistical Analysis Results presented in this work are the mean of two independent experiments. Comparison between groups was performed by the Tukey test for multiple comparisons using Statistical Package for the Social Sciences (SPSS). Statistical signicance was set at a p-value of p < 0.05. RESULTS AND DISCUSSION Operating Conditions of the Spray Dryer The pH of the feed showed no noticeable inuence on the product yield or moisture at the end of the operation, reaching in all cases an average of 35% and 4.6% in yield and moisture, respectively. For this reason pH was kept constant around pH 6.0 in the rest of the experiments. Use of 20 or 30% w=w in feed solid content resulted in no signicant difference in yield (36.1% and 36.8%, respectively) or moisture (3.9% and 4.0%, respectively). In contrast, the use of 40% w=w or 50% resulted in a negative effect in both cases. The choice of 20% w=w was made mainly because at this concentration no problems were seen during preparation, or in the passage of the emulsion through the spray nozzle, which were observed with more concentrated feeds. A similar behavior was reported

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recently by Yu et al.,[30] where the best results were obtained using feed solid contents of 20% in microencapsulation by spray drying of phospholipids. In their study, more concentrated suspensions were related to higher viscosities and difculties in atomization. Concerning biological properties of the product, pH and feed solid content showed no inuence on the viability immediately after drying. Atomizing pressure (P) has a direct effect on the diameter and cohesiveness of the particles, which in turn has a direct impact on the performance of the operation and separation in the cyclone.[16] We found that P had no inuence on moisture (average of 4.6%), a nding that disagrees with the results of Telang and Thorat, who found that increasing the pressure resulted in a product with low moisture content.[16] Atomization pressure has an important inuence on product yield. When P 1 bar, many of the drops generated were larger in size, and this situation caused them to not be totally dried in the chamber and, additionally, partially dried product stuck to the chamber walls, reducing signicantly the yield (4.4%). On the other hand, at P 3 bar, smaller particles were generated which were easily swept by the moist air, leaving the cyclone (product yield of 18.9%). Viability of the microorganism was not affected for P between 2 and 3 bar; however, use of 1 bar cannot be recommended due to the problems described above, as can be seen in Fig. 1. Taking into account the good performance in yield (26.3%) and survival (around 95%), subsequent assays were performed at P 2 bar, in

agreement with similar results obtained by Telang and Thorat[16] and Menshutina.[10] Outlet temperature (Tout) principally had an effect on nal product moisture and cell viability, but not on the yield (average of 43.8%). In Fig. 1 it is possible to see that there is a marked loss of viability as the outlet temperature rises; this effect has been reported previously,[12,2729,34] is related to drastic perturbations caused by the rate at which water is removed from cells, and results in damage to critical cell components, such as DNA and RNA.[12] Considering that it is important to keep the viability of the microorganism above 107 CFU=g,[35] at least at the beginning of storage, subsequent experiments were performed at 70 C. Lower temperatures are not recommended because it is necessary to keep the moisture below 5% in order to assure a long shelf-life of the product.[10,16] Spray Drying of Osmotically Unstressed Bacteria Figure 2 shows the effects of spray drying on osmotically unstressed bacteria. In all assays the initial count before drying was higher than 108 CFU=ml, as recommended in the literature.[36] From Fig. 2 is possible to see that the use of protective materials is absolutely needed because, in samples that were not protected, viability diminished to between 1.8 to 3.0 log cycles (more than 98%), but with the use of protective materials loss of viability was in the range of 0.27 to 0.86 log cycles (between 44 to 83%), using whey or the WMP mixture, respectively. From this it is clear that incorporation of a protective substances before drying has a positive effect, due principally to stabilization of cell

FIG. 1. Effect of atomizing pressure (.) and outlet temperature (&) in survival of autochthonous Lactobacillus plantarum immediately after drying. N: viable bacteria immediately after drying, N0: viable bacteria before drying.

FIG. 2. Survival percentage of Lactobacillus plantarum before drying (BD) immediately after drying (AD) and evaluation of cell damage (CD) in presence of various protective materials. W Whey; WM Whey Maltodextrine; SM Skim milk Maltodextrine; WMG Whey Maltodextrine Arabic gum; WMP Whey Maltodextrine Pectin; C25 Control at 25 C; C32 Control at 32 C.

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membranes and the outer coatings of the encapsulant, especially when this is made up of proteins and=or sugars.[36] Menshutina[10] also points out that the addition of protective media improved not only the survival of the microorganism, but also the ow rate of the product. Contrary to the report by Desmond,[7] for spray drying of L. paracasei, grown in a mixture of skimmed milk (10% w=v and arabic gum 10% w=v), use of arabic gum as part of the protective media did not show an impact on results after drying, compared to the other protective agents. However, arabic gum was not excluded from the storage analysis because it has been found that there is not always an agreement of results between immediate drying and after storage.[34,37] Use of a higher temperature during culture seems to provide additional protection to bacteria compared to those that were cultured at 25 C (see 25 C and 32 C bars in Fig. 2), this could be due, as already reported, to microorganisms grown at 32 C suffering mild heat stress, which increases resistance to thermal stress prior to drying.[22] This resistance is related to an adaptive response associated with increased expression of the protein GroE.[27] Results of tests of sensitivity to NaCl showed that although there was a trend for differences (AD versus CD bars in Fig. 2) between different encapsulating materials, these were not statistically signicant (p 0.279). More notable differences were seen between unprotected bacteria cultured at 25 C and 32 C, the latter showing less damage, and subsequent observation conrmed the fact that bacteria grown at higher temperature were more resistant than those cultured under normal temperature conditions; however, this protection was not comparable to the use of protective agents. Compared to results obtained by Gardiner[24] for L. salivarius and L. paracasei, using skimmed milk as the protective agent, percentages of sensitivity to NaCl in this study did not exceed 21%, whereas Gardiner saw between 70% and 100% sensitivity. Notwithstanding this, and as was noticed in the work of Gardiner, the size of colonies on MRS-NaCl plates were clearly smaller than those observed on traditional MRS plates. The fact that cell membrane damage was minimized by the use of these protective substances was a very positive result, due to the fact that this is the component of the microorganism that ultimately regulates adhesion and colonization of a hosts tissue or organ. If bacteria are not able to colonize, then they may not be able to exercise their probiotic role.[28,29] Statistical analysis considering both viability immediately after drying and cell membrane damage indicated no statistically signicant differences in the use of protective solutions of W, WM, or SM (p 0.169). The next best protective solutions were mixtures WMG and WMP (p 0.369).

Spray Drying of Osmotically Stressed Bacteria After exposure of Lactobacillus plantarum to different concentrations of NaCl during culture, it was noticed that, independent of the saline stress used, there were no signicant differences in bacterial counts at the end of exposure. This is consistent with Glaasker,[26] who found that only above a saline stress of 0.8 M in culture medium were alterations in growth of L. plantarum ATCC 14917 observed. Differing results from Kets[20] established 1.5 M as the acceptable limit for growth of L. plantarum P743. Differences between protective agents were observed only after drying, where loss of viability caused by the process was always higher in samples that had been osmotically stressed. Such behavior was more noticeable as the level of salt stress applied rose. This observation drew our attention because, under similar conditions, the Lactobacillus acidophilus strain showed greater resistance during spray drying when a saline stress of 0.25 M was used during batch fermentation.[28] Also, Lactobacillus plantarum showed good resistance to drying when using a saline concentration of 1.0 M during culture[20]; however, this latter result is not entirely comparable to that which we have performed in this work, because drying was carried out in a much less aggressive way than usually is involved in spray drying. According to Csonka,[25] bacteria respond to osmotic stress by the accumulation of certain solutes, termed compatibles because their excess does not negatively affect the microorganism. The function of these solutes is to restore the osmotic balance by mean water passage through the cell membrane in order to avoid hyper or hypo-osmotic shock. Proposed compatible solutes include: potassium ions, sugars, polyols, amino acids, and quaternary amines. In the specic case of lactic acid bacteria, accumulation of one or more of these compatible solutes is dependent on the species.[22] L. plantarum has been found to be unable to respond adequately to osmotic stress caused by the accumulation of sodium or potassium ions, which have an immediate effect on the swelling of the cell membrane and a negative effect on macromolecular components of the cytoplasm.[20] Additionally, alterations in fatty acid composition of the cell membrane have been also reported, which will clearly affect transport through it.[38,39] In summary, if during salt stress the most affected component in L. plantarum is the cell membrane, as would be expected during spray drying where it is the most exposed zone to dehydration, bacterial viability will be more affected as they had been exposed to a higher level of salinity during culture, all of which is consistent with our experimental results. The effect of protective substances was also analyzed statistically; results suggested that there were no differences when using W, WM, or SM. Use of small amounts of arabic gum (WMG) or pectin (WMP) showed a marked loss of viability as the level of stress was increased. Additionally,

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under a stress of 0.75 M we observed a slight increase in cell damage, in comparison to other protective suspensions. Bacteria cultured at 32 C further showed the effects of saline stress during spray drying, with a steady reduction in viability, as well a marked effect on cell membrane damage immediately after drying, highlighting the importance of using protective materials. Independent of the fact that some protective materials were more effective than others, and that use of osmotic stress weakened the resistance of bacteria during drying, the count of viable microorganism immediately after drying was in the range 7.3 109 to 3.5 108 CFU=g. Considering that for probiotic purposes a count of viable bacteria must be between 106 to 108 CFU=g,[6,8,34,35] all substances and mixtures assayed would serve this purpose, at least immediately after drying. Finally, although it was noticed that the best results were obtained without osmotic stress, this statement does not imply that bacteria were not stressed at all. It is important to remember that the bacteria had been harvested during the stationary phase of growth, during which they were exposed to substrate competition and inhibition by the presence of metabolites (e.g., lactic acid) and that these situations in themselves create additional resistance to drying, in comparison to bacteria that have been harvested during the exponential growth phase, and in which resistance has been enhanced through the use of a sub-lethal stress prior to drying.[9,21,40] In fact, as pointed out by van de Guchte, bacteria in the stationary phase of growth develop an overall resistance to stress.[22] Storage The results of storage of the dehydrated product are shown in Figs. 3A3E. As has been pointed by Li et al.,[9] cell injuries occur not only during processing but also during storage of dried cultures. In our case, during storage, there was a gradual decrease in viability of L. plantarum in the dehydrated product. The degree of this decrease was most clearly inuenced by the protective material. Statistical analysis showed that samples of osmotically unstressed bacteria showed better survival than those that had been osmotically stressed. After 70 days of sampling, cheese whey (W) was the substance which gave the best results in terms of stability of the strain, achieving a viability of 9.98 0.04 log CFU=g. In a separated group were the mixtures WM and SM, while in the third and fourth position were samples containing pectin and gum, respectively. In the latter cases, the concentration of viable microorganism fell below 108 CFU=g after six weeks of storage. The aim of a protective agent is mainly to coat the bacteria during the drying process in order to avoid high

temperatures that can cause its immediate death. The way in which protective materials function is related principally to their ability to stabilize cell membranes, as this is where water molecules are lost during dehydration,[40] but they also function as antioxidants and anti-moisture agents during shelf-life.[36] Considering these functions, it has been found that disaccharides act better than polysaccharides. This is because polysaccharides tend to have difculty interacting with the cell membrane because of their larger size, and therefore the protection provided by these sugars is lower. In this work, using maltodextrin, arabic gum, or pectin (all polysaccharides), steric problem became more noticeable as osmotic stress was increased, probably in these cases due to the membrane having some sensitivity even before drying. Cheese whey is composed principally of lactose, a disaccharide which has been shown to be able to protect bacteria especially during freeze drying.[41] Once water molecules are removed, disaccharides are capable of forming hydrogen bonds with proteins in the cell membrane, which helps to prevent their denaturation and, with this, helps prevent total rupture of the membrane.[9,4143] Results obtained in this study corroborate existing reports, since in those samples in which only whey was used as protectant, survival was greater than 90%, even after 10 weeks of storage. Use of cheese whey instead of lactose has the advantage that it contains lipids and proteins which give an additional protection to bacteria. Also, there is evidence that the presence of small quantities of whey (less than 5%) can improve the microcapsules, increasing the elasticity of the protectant lm formed during spray drying and helping to avoid cracked particles, and hence protecting the cells inside them.[44] In this work, it was also postulated that the use of whey may decrease the amount of contact adhesion between two particles, or between a particle and a surface, so it may have been responsible for the improved product yield in comparison to the use of pure lactose, which generated both cracked microcapsules and those with a smooth surfaces. It should be noted that further improvements can be made in terms of storage of the product, as the importance of environmental conditions in terms of temperature and relative humidity has been reported,[45] along with suitable packaging, as a way to prevent the passage of light, moisture, and=or oxygen.[34] It is important to note that, contrary to that reported by Oliveira,[37] in relation to limitations of spray drying on the stabilization of probiotic bacteria, no signicant differences in the count of viable microorganisms after 60 days of storage at 4 C were found, although in Oliveiras work using the Lactobacillus acidophilus strain, a much more complex technique of protection (complex coacervation) was used, and the samples were dried at a lower temperature (46 C vs. 70 C) in a bed dryer.

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FIG. 3. (A) Viability of Lactobacillus plantarum during storage at 4 C in presence of whey (W) and under different conditions of osmotic stress during culture. C 25 is referred to osmotically unstressed culture at 25 C dried without protection; (B) viability of Lactobacillus plantarum during storage at 4 C in presence of whey maltodextrine (WM) and under different conditions of osmotic stress during culture. C 25 is referred to osmotically unstressed culture at 25 C dried without protection; (C) viability of Lactobacillus plantarum during storage at 4 C in presence of skim milk maltodextrine (SM) and under different conditions of osmotic stress during culture. C 25 is referred to osmotically unstressed culture at 25 C dried without protection; (D) viability of Lactobacillus plantarum during storage at 4 C in presence of whey maltodextrine arabic gum (WMG) and under different conditions of osmotic stress during culture. C 25 is referred to osmotically unstressed culture at 25 C dried without protection; (E) viability of Lactobacillus plantarum during storage at 4 C in presence of whey maltodextrine pectin (WMP) and under different conditions of osmotic stress during culture. C 25 is referred to osmotically unstressed culture at 25 C dried without protection.

Growth of Dried Autochthonous Lactobacillus plantarum Once bacteria had been stabilized, it was necessary to evaluate whether they maintain the same characteristics and abilities in comparison to a fresh strain (coming from stock culture).[46] Figures 4A and 4B show growth and acidication kinetics of autochthonous Lactobacillus plantarum followed over a period of 48 hours. According

to the best results previously obtained, osmotically unstressed bacteria protected with cheese whey (20% w=w) were tested. The rst thing that is notable in Figs. 4A and 4B is that the behavior of both strains is almost the same. In both cases, the stationary phase of growth was reached after around 15 hours, and the nal concentrations of viable microorganism was 2.9 109 CFU=ml. A strong variation

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FIG. 5. Growth kinetic of Yersinia ruckeri in presence of autochthonous Lactobacillus plantarum in fresh and dried form (P 2 bar, Tout 70 C, Protective material Whey, Osmotically unstressed bacteria).

FIG. 4. (A) Growth kinetic of Lactobacillus plantarum in fresh and dried form stabilized by cheese whey (20% w=w) and cultured without osmotic stress; (B) acidication kinetic of Lactobacillus plantarum in fresh and dried form stabilized by cheese whey (20% w=w) and cultured without osmotic stress.

in pH was also detected at the same period for fresh and dehydrated strains. During the exponential growth phase (612 h), maximum growth rates (lm) obtained using the Gompertz model were 3.2 and 2.1 h1 for fresh and dehydrated L. plantarum, respectively. The difference could be related to the time that dried microorganisms have to spend rehydrating and adapting after their latency state. Even so, viability at the end of culturing demonstrates the effectiveness of the protection afforded by whey during and after drying. Inhibition of Yersinia ruckeri According to Ringo et al.,[2] there is strong in vitro and in vivo evidence concerning the effectiveness of treatments in shes with LAB in the presence of pathogens such as Vibrio anguillarum, Aeromonas salmonicida, or Yersinia ruckeri. However, in all assays performed, cells used were

of the fresh form. In relation to this, Gatesoupe stated his doubts concerning the feasibility of using inactivated cells for this purpose, namely because the protection that they could provide would be limited compared to that provided by active cells.[4] In our work, once growth of the dehydrated strain had been veried, it was necessary to evaluate whether probiotic ability had been altered in any way. In the specic case of the strain used in this study, its probiotic strength is related to its high acidifying capacity, which enables it to inhibit growth of pathogens. This inhibitory behavior is shown in Fig. 5, and as can be seen, growth of Yersinia was clearly affected by the probiotic strain, either in dried or fresh forms. After a period of 48 hours, growth of Yersinia ruckeri in culture without Lactobacillus plantarum achieved a concentration of 7.4 109 CFU=ml; however, in samples where L. plantarum was present, growth of the pathogen was diminished to 1.8 log cycles. These results are an important rst step to answering the question presented by Gatesoupe[4] concerning the health benets given by live probiotics versus those conferred by inactivated cells. In order to evaluate its effectiveness, in vivo assays are currently being performed to investigate its inclusion in sh feed formulation. CONCLUSIONS We found that it is possible to obtain a dried product of the same characteristics, in terms of growth and probiotic ability, as that of a fresh strain, but which did not have the limitations associated with this kind of product, such as transport difculties, the imminent risk of contamination, or lost of viability caused by mishandling.

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Concerning operating conditions of the spray dryer, a feed solid content of 20% with an atomizing pressure of 2 bar and an outlet temperature of 70 C gave the best results. Also, it was found that pH of the feed had no noticeable effect on the product yield or moisture. Once the best operating conditions of the spray dryer were found, it was possible to obtain a product with a high viability of microorganism ($109 CFU=g), using osmotically unstressed bacteria and cheese whey (20% w=w) as the protective agent during spray drying. This viability was almost unchanged after 10 weeks of storage at 4 C under non-controlled atmospheric conditions. In addition, the dried strain was able to grow and inhibit a pathogen in the same way as a fresh strain (around 2 log cycles). As was shown, and contrary to expectations, use of salt stress during growth did not have any benet to the resistance of native Lactobacilllus plantarum to spray drying. In fact, as saline stress increased, survival after spray drying decreased. This behavior was more noticeable in those samples that were dried in the presence of small quantities of arabic gum or pectin. Thermally stressed bacteria (grown at 32 C) showed more resistance to drying than those that were not thermally stressed (1% versus 0.01%), even though the protection afforded by this type of stress was not comparable to that provided by encapsulation materials. It would be recommendable in the future to investigate whether a combination of both procedures was able to further improve viability after drying. Finally, results obtained in this study point to the feasibility of obtaining a stable, highly viable, and probiotic product using microencapsulation by spray drying. ACKNOWLEDGMENTS The authors wish to thank Project INNOVA CHILE 05CT6PPT-13 and Project FONDECYT 1060919 for nancial support. REFERENCES
1. SalmonChile Report. Summary of the Chilean salmon industry (N 1= 2008) Available at http://www.salmonchile.cl/frontend/index.asp. 2008. Last accessed on January 2012. 2. Ringo, E.; Lovmo, L.; Kristiansen, M.; Bakken, Y.; Salinas, I.; Myklebust, R.; Olsen, R.E.; Mayhew, T.M. Lactic acid bacteria vs. pathogens in the gastrointestinal tract of sh: A review. Aquaculture Research 2010, 41, 451467. 3. Cabello, F.C. Heavy use of prophylactic antibiotics in aquaculture: A growing problem for human and animal health and for the environment. Environmental Microbiology 2006, 8(7), 11371144. 4. Gatesoupe, F.J. Updating the importance of lactic acid bacteria in sh farming: Natural occurrence and probiotic treatments. Journal of Molecular Microbiology and Biotechnology 2007, 14, 107114. 5. Kesarcodi-Watson, A.; Kaspar, H.; Lategan, M.J.; Gibson, L. Probiotics in aquaculture: The need, principles and mechanism of action and screening processes. Aquaculture 2008, 274, 114.

rquez, R. Drying and storage stability of 6. Toledo, N.; Ferrer, J.; Bo a probiotic strain incorporated into a sh feed formulation. Drying Technology 2010, 28, 508516. 7. Desmond, C.; Ross, R.P.; OCallaghan, E.O.; Fitzgerald, G.; Stanton, C. Improved survival of Lactobacillus paracasei NFBC 338 in spray-dried powders containing gum acacia. Journal of Applied Microbiology 2002, 93, 10031011. 8. Peighambardoust, S.H.; Tafti, A.G.; Hesari, J. Application of spray drying for preservation of lactic acid starter cultures: A review. Trends in Food Science and Technology 2011, 22(5), 215224. 9. Li, X.Y.; Chen, X.G.; Liu, C.S.; Peng, H.N.; Cha, D.S. Effect of trehalose and drying process on the survival of encapsulated Lactobacillus casei ATCC 393. Drying Technology 2008, 26, 895901. 10. Menshutina, N.V.; Gordienko, M.G.; Voinovskiy, A.A.; Zbicinski, I. 2010. Spray drying of probiotics: Process development and scale-up. Drying Technology 2010, 28, 11701177. 11. Desai, K.G.H.; Park, H.J. Recent developments in microencapsulation of food ingredients. Drying Technology 2005, 23, 13611394. 12. Varsha, S.; Bhaskar, T. Formulation and cost-effective drying of probiotic yeast. Drying Technology 2011, 29, 749757. 13. Corcoran, B.M.; Ross, R.P., Fitzgerald, G.F.; Stanton, C. Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances. Journal of Applied Microbiology 2004, 96, 10241039. 14. Santivarangkna, C.; Kulozik, U.; Foerst, P. Inactivation mechanism of lactic acid starter cultures preserved by drying processes. Journal of Applied Microbiology 2008, 105, 113. , M.I. Microencapsulation by spray drying. Drying Technology 15. Re 1998, 16(6), 11951236. 16. Telang, A.M.; Thorat, B.N. Optimization of process parameters for spray drying of fermented soy milk. Drying Technology 2010, 28, 14101456. 17. Gibbs, B.F.; Kermasha, S.; Alli, I.; Mulligan, C. Encapsulation in the food industry: A review. International Journal of Food Sciences and Nutrition 1999, 50(3), 213224. 18. Shiga, H.; Yoshii, H.; Nishiyama, T.; Furuta, T.; Forssele, P.; Poutanen, K.; Linko, P. Flavor encapsulation and release characteristics of spray-dried powder by the blended encapsulant cyclodextrin and gum arabic. Drying Technology 2001, 19(7), 13851395. 19. Anal, A.K.; Singh, H. Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends in Food Science and Technology 2007, 18, 240251. 20. Kets, E.P.W.; Teunissen, P.J.M.; Bont, J.A.M. Effect of compatible solutes on survival of lactic acid bacteria subjected to drying. Applied and Environmental Microbiology 1996, 62(1), 259261. 21. Kim, W.S.; Perl, L.; Park, J.H.; Tandianus, J.E.; Dunn, N.W. Assessment of stress response of the probiotic Lactobacillus acidophilus. Current Microbiology 2001, 43, 346350. 22. Van de Guchte, M.; Serror, P.; Chervaux, C.; Smokvina, T.; Ehrlich, n, E. Stress responses in lactic acid bacteria. Antonie van S.D.; Mag Leeuwenhoek 2002, 82, 187216. 23. Lorca, G.L.; Raya, R.R.; Taranto, M.P.; de Valdez, G.F. Adaptative acid tolerant response in Lactobacillus acidophilus. Biotechnology Letters 1998, 20(3), 239241. 24. Gardiner, G.; Sullivan, E.; Nelly, J.; Auty, A.; Fitzgerald, G.; Collins, J.; Ross, R.; Stanton, C. Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray dying. Applied Environmental Microbiology 2000, 66(6), 26052612. 25. Czonka, L. Physiological and genetic responses of bacteria to osmotic stress. Microbiological Reviews 1989, 53(1), 121147. 26. Glaasker, E.; Tjan, F.S.; Ter Steeg, P.F.; Konings, W.N.; Poolman, B. Physiological response of Lactobacillus plantarum to salt and nonelectrolyte stress. Journal of Bacteriology 1998, 180(17), 47184723.

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RQUEZ BUSTOS AND BO followed by spouted-bed drying. Drying Technology 2007, 25(10), 16871693. Guillot, A.; Obis, D.; Mistou, M.Y. Fatty acid membrane composition and activation of glycine-betaine transport in Lactococcus lactis subjected to osmotic stress. International Journal of Food Microbiology 2000, 55(3), 4751. Guerzoni, M.E.; Lanciotti, R.; Cocconcelli, S. Alteration in cellular fatty acid composition as a response to salt, acid, oxidative and thermal stresses in Lactobacillus helveticus. Microbiology 2001, 147, 22552264. Teixeira, P.C.; Castro, M.H.; Kirby, R.M. Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus. Journal of Applied Bacteriology 1995, 78(4), 456462. Leslie, S.B.; Israeli, E.; Lighthart, B.; Crowe, J.H.; Crowe, L.M. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and Environmental Microbiology 1995, 61(10), 35923597. Crowe, J.H.; Crowe, L.M.; Carpenter, F.; Aurell, C. Stabilization of dry phospholipids bilayers and proteins by sugars. Biochemical Journal 1987, 242, 110. Martos, G.I.; Minahk, C.J.; Font de Valdez, G.; Morero, R. Effects of protective agents on membrane uidity of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus. Letters in Applied Microbiology 2007, 45, 282288. Wang, S.; Langrish, T. The use of surface active compounds as additives in spray drying. Drying Technology 2010, 28, 341348. Golowczyc, M.A.; Gerez, C.L.; Silva, J.; Abraham, A.G.; De Antoni, G.L.; Teixeira, P. Survival of spray-dried Lactobacillus ker is affected by different protectants and storage conditions. Biotechnology Letters 2010, 33(4), 681686. Ananta, E.; Volkert, M.; Knorr, D. Cellular injuries and storage stability of spray-dried Lactobacillus rhamnosus GG. International Dairy Journal 2005, 15, 399409.

Downloaded by [Universidad De Concepcion] at 11:50 10 January 2013

27. Silva, J.; Carvalho, A.S.; Pereira, H.; Teixeira, P.; Gibas, P.A. Induction of stress tolerance in Lactobacillus delbrueckii ssp. bulgaricus by the addition of sucrose to the growth medium. Journal of Dairy Research 2004, 71, 121125. 28. Riveros, B.; Ferrer, J.; Borquez, R. Spray drying of a vaginal probiotic strain of Lactobacillus acidophilus. Drying Technology 2009, 27, 123132. 29. Meng, X.C.; Stanton, C.; Fitzgerald, G.F.; Daly, C.; Ross, R.P. Anhydrobiotics: The challenges of drying probiotic cultures. Food Chemistry 2008, 106, 14061416. 30. Yu, C.; Wang, W.; Yao, H.; Liu, H. Preparation of phospholipid microcapsule by spray drying. Drying Technology 2007, 25, 695702. 31. Silva, J.; Freixo, R.; Gibbs, P.; Teixeira, P. Spray-drying for the production of dried cultures. International Journal of Dairy Technology 2011, 64(3), 321335. 32. Association of Ofcial Analytical Chemists. Ofcial Methods of Analysis of the Association of Ofcial Analytical Chemists, 17th ed.; AOAC International: Arlington, VA, 2000. 33. Zwietering, M.H.; Jongenburger, I.; Rombouts, F.M.; Vant Riet, K. Modeling of the bacterial growth curve. Applied Environmental Microbiology 1989, 56(6), 121147. vez, B.E.; Ledeboer, A.M. Drying of probiotics: Optimization 34. Cha of formulation and process to enhance storage survival. Drying Technology 2007, 25, 11931201. 35. Li, X.; Lin, S.X.; Chen, X.; Chen, L.; Pearce, D. Inactivation kinetics of probiotic bacteria during the drying of single milk droplets. Drying Technology 2006, 24, 695701. 36. Morgan, C.A.; Herman, N.; White, P.A.; Vesey, G. Preservation of micro-organism by drying: A review. Journal of Microbiological Methods 2006, 66, 183193. 37. Oliveira, A.C.; Moretti, T.S.; Boschini, C.; Baliero, J.C.C.; Freitas, L.A.P.; Freitas, O.; Favaro-Trindade, C.S. Microencapsulation of B. lactis (Bl 01) and L. acidophilus (LAC 4) by complex coacervation

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