Encapsulation of Lactobacillus acidophilus La-5 and Bifidobacterium Bb-12 by spray drying and evaluation of its resistance in simulated gastrointestinal conditions, thermal treatments and storage conditions

Encapsulação de Lactobacillus acidophilus La-5 e Bifidobacterium Bb-12 por spray dryer e avaliação da sua resistência em condições gastrointestinais simuladas, tratamentos térmicos e em condições de armazenamento

Graciele Lorenzoni Nunes Mariana Heldt Motta Alexandre José Cichoski Roger Wagner Édson Irineu Muller Cristiane Franco Codevilla Cristiane de Bona da Silva Cristiano Ragagnin de Menezes About the authors

ABSTRACT:

Lactobacillus acidophillus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles were produced at different temperatures by spray dryer. The influence of different temperatures on the viability, encapsulation efficiency, water activity and moisture were evaluated. Microparticles that presented more viability were submitted to thermal resistance, gastrointestinal simulation, storage stability, morphology and particle size analyses. Drying temperature of 130°C showed higher encapsulation efficiency, 84.61 and 79.73% for Lactobacillus acidophillus (ML) and Bifidobacterium Bb-12 (MB) microparticles, respectively. In the evaluation of thermal resistance and gastrointestinal simulation, the microparticles of Lactobacillus acidophillus La-5 (ML) presented higher survival than Bifidobacterium Bb-12 (MB) under these conditions. In storage viability only the Lactobacillus acidophillus La-5 (ML) microparticles remained viable at all evaluated temperatures during the 120 days. The particle sizes reported were 4.85 for Lactobacillus acidophillus La-5 (ML) and 8.75 for Bifidobacterium Bb-12 (MB), being in agreement with the desired values for products obtained by spray dryer. Finally, the Lactobacillus acidophilus La-5 (ML) microparticles were shown to be more resistant under the conditions evaluated in this study.

Key words:
spray dryer; viability; probiotics

RESUMO:

Micropartículas de Lactobacillus acidophillus La-5 (ML) e Bifidobacterium Bb-12 (MB) foram produzidas em diferentes temperaturas de secagem no spray dryer. A influência das diferentes temperaturas sobre a viabilidade, eficiência de encapsulação, atividade de água e umidade foram avaliadas. As micropartículas que apresentaram maior viabilidade foram submetidas a análises de resistência térmica, simulação gastrointestinal, estabilidade ao armazenamento, morfologia e tamanho de partícula. A temperatura de secagem de 130°C mostrou maior eficiência de encapsulação, 84.61 e 79.73% para micropartículas de Lactobacillus acidophillus (ML) e Bifidobacterium Bb-12 (MB), respectivamente. Na avaliação da resistência térmica e simulação gastrointestinal as micropartículas de Lactobacillus acidophillus La-5 (ML) apresentaram maior sobrevivência que Bifidobacterium Bb-12 (MB) nestas condições. Na viabilidade ao armazenamento somente as micropartículas Lactobacillus acidophillus La-5 (ML) mantiveram-se viáveis em todas as temperaturas avaliadas durante os 120 dias. Os tamanhos de partícula encontrados foram de 4.85 para Lactobacillus acidophillus La-5 (ML) e 8.75 para Bifidobacterium Bb-12 (MB), estando de acordo aos valores desejáveis para produtos obtidos por spray dryer. Por fim, as micropartículas de Lactobacillus acidophilus La-5 (ML) demostraram ser mais resistentes frente as condições avaliadas neste estudo.

Palavras-chave:
spray dryer; viabilidade; probióticos

INTRODUCTION:

Probiotics are live microorganisms, which when consumed in adequate amounts, conferring a health benefit for the host. In this context, in recent years there has been a great deal of interest in its use and application in food especially in fermented dairy products (yogurt, fermented milk and cheese). However, there is also increasing interest for the use of probiotics in non-dairy products such as fruit and vegetable juices, soy and some cereals (FAO/OMS, 2001; DAS & GOYAL, 2015DAS, D.; GOYAL, A. Antioxidant activity and g-aminobutyric acid (GABA) producing ability of probiotic Lactobacillus plantarum DM5 isolated from Marcha of Sikkim. LWT-Food Science and Technology, v. 61, p. 263-268, 2015. Available from: http://www.sciencedirect.com/science/article/pii/S0023643814007166>. Accessed: Nov. 08, 2017. doi: 10.1016/j.lwt.2014.11.013.
http://www.sciencedirect.com/science/art...
; FIJAN, 2014FIJAN, S. 2014. Microorganisms with claimed probiotic properties: An overview of recent literature. International Journal of Environmental Research and Public Health, v. 11, p. 4745-4767. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053917/>. Accessed: Dec. 07, 2017. doi: 10.3390/ijerph110504745.
https://www.ncbi.nlm.nih.gov/pmc/article...
; RANADHEERA et al., 2017RANADHEERA et al. Probiotic delivery through fermentation: dairy vs. non-dairy everages. Fermentation, v. 3. p. 1-17, 2017. Available from: http://www.mdpi.com/2311-5637/3/4/67>. Accessed: Apr. 11, 2018. doi: 10.3390/fermentation3040067.
http://www.mdpi.com/2311-5637/3/4/67...
). Bifidobacterium lactis and Lactobacillus acidophillus are the probiotic bacteria most widely studied and frequently used in food (FELICIO et al., 2016FELICIO, T. L. et al. Physico-chemical changes during storage and sensory acceptance of low sodium probiotic minas cheese added with arginine. Food Chemistry, v. 196, p. 628-637, 2016. Available from: https://www.ncbi.nlm.nih.gov/pubmed/26593536>. Accessed: Dec. 11, 2017. doi: 10.1016/j.foodchem.2015.09.102.
https://www.ncbi.nlm.nih.gov/pubmed/2659...
; HOMAYOUNI et al., 2008HOMAYOUNI, A. et al. Effect of microencapsulation and resistant starch on the probiotic survival and sensory properties of synbiotic ice cream. Food Chemistry, v. 111, p. 50-55, 2008. Available from: https://www.sciencedirect.com/science/article/pii/S0308814608003245>. Accessed: Dec. 02, 2017. doi: 10.1016/j.foodchem.2008.03.036.
https://www.sciencedirect.com/science/ar...
). RANADHEERA et al. (2015RANADHEERA et al. Microencapulation of Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. Lactis BB-12 and Propionibacterium jansenii 702 by spray drying in goast’s milk. Small Ruminant Research, v. 123, p. 155-159, 2015. Available from: https://www.sciencedirect.com/science/article/pii/S0921448814003058>. Accessed: Apr. 11, 2018. doi: 10.1016/j.smallrumres.2014.10.012.
https://www.sciencedirect.com/science/ar...
) microencapsulated Lactobacillus acidophilus La-5, Bifidobacterium Bb-12 and Propionibacterium jansenii 702 by spray drying in goat’s milk. Recently, Kavitek et al. (2018KAVITEK et al. Recent developments on encapsulation of lactic acid bacteria as potential starter culture in fermented foods - A review. Food Bioscience, v. 21, p. 34-44, 2018. Available from: https://www.sciencedirect.com/science/article/pii/S2212429217302961>. Accessed: Apr. 11, 2018.
https://www.sciencedirect.com/science/ar...
) demonstrated recent developments on encapsulation of lactic acid bacteria as potential starter culture in fermented foods. According to ANAL & SINGH (2007ANAL, A. K.; SINGH, H. Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends in Food Science & Technology, v. 18, p. 240-251, 2007. Available from: https://www.sciencedirect.com/science/article/pii/S0924224407000350>. Accessed: Nov. 03, 2017. doi: 10.1016/j.tifs.2007.01.004.
https://www.sciencedirect.com/science/ar...
), the ability of probiotic microorganisms to survive and develop in the host will directly influence their probiotic effects. Therefore, the microorganism that is metabolically stable in the product and survive the passage through the gastrointestinal tract reaching the intestine with high viability will be able to develop its beneficial effects. Application of microencapsulation processes has been studied as an alternative to maintain high viability of these microorganisms (FUNG et al, 2011FUNG, W. Y. et al. Agrowaste-based nanofibers as a probiotic encapsulant: fabrication and characterization. Journal of Agricultural and Food Chemistry, v. 59, p. 8140-8147, 2011. Available from: https://www.ncbi.nlm.nih.gov/pubmed/21711050>. Accessed: Nov. 12, 2017. doi: 10.1021/jf2009342.
https://www.ncbi.nlm.nih.gov/pubmed/2171...
; KIM et al., 2011). Among different microencapsulation techniques, spray drying is commonly used for its advantages such as low operating costs, high production rates, low moisture content in the final product and possibility of application on an industrial scale (CORCORAN et al., 2004CORCORAN, B. M., et al. Comparative survival of probiotic lactobacilli spray dried in the presence of prebiotic substances. Journal of Applied Microbiology, v, 96, p. 1024-1039, 2004. Availabe from: < Availabe from: https://www.ncbi.nlm.nih.gov/pubmed/15078519 >. Accessed: Nov. 13, 2016. doi: 10.1111/j.1365-2672.2004.02219.x.
https://www.ncbi.nlm.nih.gov/pubmed/1507...
). However, this process requires high temperatures, which may affect the survival of probiotic microorganisms (BOZA et al., 2004BOZA, Y. et al. Effect of spray-drying on the quality of encapsulated cells of Beijerinckia sp. Process Biochemistry, v. 39, p. 1275-1284, 2004. Available from: https://www.sciencedirect.com/science/article/pii/S0032959203002577>. Accessed: Oct. 25, 2017. doi: 10.1016/j.procbio.2003.06.002.
https://www.sciencedirect.com/science/ar...
). In this sense, optimization of the spray-drying conditions as well as the composition of the encapsulation solution are parameters of great importance in order to achieve high survival of the probiotic microorganisms during this process (CORCORAN et al., 2004CORCORAN, B. M., et al. Comparative survival of probiotic lactobacilli spray dried in the presence of prebiotic substances. Journal of Applied Microbiology, v, 96, p. 1024-1039, 2004. Availabe from: < Availabe from: https://www.ncbi.nlm.nih.gov/pubmed/15078519 >. Accessed: Nov. 13, 2016. doi: 10.1111/j.1365-2672.2004.02219.x.
https://www.ncbi.nlm.nih.gov/pubmed/1507...
; FRITZEN-FREIRE et al., 2012FRITZEN-FREIRE, C. B. et al. Microencapsulation of bifidobacteria by spray drying in the presence of prebiotics. Food Research International, v. 45, p. 306-312, 2012. Available from: https://www.sciencedirect.com/science/article/pii/S0963996911005539>. Accessed: Nov. 18, 2017. doi: 10.1016/j.foodres.2011.09.020.
https://www.sciencedirect.com/science/ar...
; RAJAM et al., 2013RAJAM, R. P. et al. Effect of whey protein-alginate wall systems on survival of microencapsulated Lacotobacillus plantarum in simulated gastrointestinal conditions. Journal of Functional Foods, v. 4, p. 891-898, 2013. Available from: https://www.sciencedirect.com/science/article/pii/S1756464612001016>. Accessed: Oct. 15, 2017. doi: 10.1016/j.jff.2012.06.006.
https://www.sciencedirect.com/science/ar...
; SIMPSON et al., 2005SIMPSON, P. J. et al. Intrinsic tolerance of Bifidobacterium species to heat and oxygen and survival following spray drying and storage. Journal of Applied Microbiology , v. 99, p. 493-501, 2005. Available from: https://www.ncbi.nlm.nih.gov/pubmed/16108790>. Accessed: Dec. 09, 2017. doi: 10.1111/j.1365-2672.2005.02648.x.
https://www.ncbi.nlm.nih.gov/pubmed/1610...
).

In this context, the aim of this study was to evaluate the influence of different temperatures by spray drying on the viability, encapsulation efficiency, water activity and moisture of microparticles containing Lactobacillus acidophillus La-5 and Bifidobacterium Bb-12. Subsequently, the microparticles that presented the highest viability and best physical-chemical characteristics were evaluated in relation to their thermal resistance, gastrointestinal simulation and storage stability. In addition, the morphology and particle size were also evaluated.

MATERIALS AND METHODS:

To produce microparticles, the following compounds were used: Gum arabic (CNI, São Paulo, Brazil); Maltodextrin (Ingredion, São Paulo, Brazil); Tween 80 (Vetec, Rio de Janeiro, Brazil); Glycerol (Vetec, Rio de Janeiro, Brazil), and probiotic culture Lactobacillus acidophilus La-5 and Bifidobacterium Bb-12 obtained by Chr. Hansen from Brazil (Valinhos, São Paulo).

Inoculum

The Lactobacillus acidophilus La-5 probiotic culture (Chr. Hansen, São Paulo, Brazil) was activated in MRS broth (Himedia Curitiba, Parana, Brazil) and incubated for 15 h at 37°C. The Bifidobacterium Bb-12 culture was rehydrated in reconstituted milk (Molico, Nestlé, São Paulo, Brazil) at a concentration of 12% and incubated for 5 hours at 37°C. Then, it was centrifuged at 4670 g for 15 min and washed in NaCl solution (0.85%). Cells were then suspended in saline to obtain a solution containing about 12 and 10log CFU/g-1.

Production of microparticles by spray drying

Feed solutions were prepared with gum arabic (8g), maltodextrin (2g), glycerol (1.9mL), tween 80 (0.1mL) containing Lactobacillus acidophilus La-5 (SL) and Bifidobacterium Bb-12 (SB) to a final concentration of 12% m/v. Microencapsulation process was performed in a lab spray dryer (MSD 1.0 Labmaq, Sao Paulo, Brazil). Initially, the feed solutions (SL and SB) were submitted to different drying temperatures, 110, 120, 130 and 140°C. Next, the microparticles produced at the inlet temperature of 130°C were chosen to be further evaluated. Different feed solutions, kept stirring, were introduced into the drying chamber using a peristaltic pump with feed rate of 0.48L/h, drying air flow rate of 40L/min, and air pressure of 0.6 MPa. The microparticles (ML and MB) were collected at the base of the cyclone, transferred to sterile vials, and stored in a desiccator.

Viable cell count

Serial dilutions for Lactobacillus acidophilus La-5 and Bifidobacterium Bb-12 were transferred to sterile Petri plates containing MRS agar (Himedia Curitiba, Paraná, Brazil), in triplicate. The MRS agar used for Bifidobacterium Bb-12 was added lithium chloride (0.1%) and L-cysteine (0.05%), according to manufacturer recommendations (Chr Hansen, 1999). Plates were incubated at 37°C for 72h in anaerobic jars with an anaerobic generator (Oxoid, São Paulo, Brazil).

Dilution of the microparticles comprised weighing 1g of microparticles followed by the addition of 9mL sterile phosphate buffer solution (pH 7.5), following the methodology described by SHEU et al. (1993SHEU, T. Y.; et al. Improving survival of culture bacteria in frozen desserts by microentrapment. Journal of Dairy Science, v. 76, p.1902-1907, 1993. Avaliable from: < Avaliable from: https://www.ncbi.nlm.nih.gov/pubmed/8345127 >. Accessed: Nov. 11, 2017. doi: 10.3168/jds.S0022-0302(93)77523-2.
https://www.ncbi.nlm.nih.gov/pubmed/8345...
). Results were shown as log colony forming units per gram (log CFU/g-1).

Efficiency of encapsulation (EE)

The efficiency of encapsulation (EE) is the survival rate of the microorganisms during the microencapsulation process, calculated according to Eq. (1), as proposed by MARTIN et al. (2013MARTIN, M. J. et al. Effect of unmodified starch on viability of alginate-encapsulated Lactobacillus fermentum CECT5716. LWT - Food Science and Technology, v. 53, p.480-486, 2013. Available from: https://www.sciencedirect.com/science/article/pii/S002364381300114X>. Accessed: Oct. 21, 2017. doi: 10.1016/j.lwt.2013.03.019.
https://www.sciencedirect.com/science/ar...
):

EE% = (N/N0) x 100 (1)

Where N is the number of viable cells (log CFU/g-1) released from the microparticles and N0 is the number of viable cells (log CFU/g-1) free in the feed solution before the spray-drying process. Viable cell count was performed as described in Section “Viable cell count”.

Moisture and Water Activity (Aw)

Moisture content of the microparticles was determined in an oven at 105°C until constant weight, according to the methodology proposed by AOAC (2005)Association of Official Analytical Chemists - AOAC. 2005. Official methods of analysis of the association analytical chemists (18th. Edition). Maryland, USA.. Water activity was measured at 25°C using Aqualab 4TE equipment (Decagon Devices, Pullman, WA, USA) after prior stabilization of the samples for 15min.

Microparticle morphology and size

Morphology of the microcaparticles was evaluated using an optical microscope (Carl Zeiss Axio Scope. A1, Oberkochen, Germany) equipped with an Axio Cam MRc digital camera (Carl Zeiss) and scanning electronic microscope (SEM; JEOL JM6360, Tokyo, Japan). Distribution of microparticle size was measured using a Mastersizer 3000 (Malvern, Germany), with water as the dispersion medium.

Resistance to heat treatment

Thermal resistance was assessed as proposed by ZHANG et al. (2015ZHANG, Y.; LIN, J. B.; ZHONG, Q. The increased viability of probiotic Lactobacillus salivarius NRRL B-30514 encapsulated in emulsions with multiple lipid-protein-pectin layers. Food Research International, v. 71, p. 9-15, 2015. Available from: https://www.sciencedirect.com/science/article/pii/S0963996915000782>. Accessed: Nov. 01, 2017. doi: 10.1016/j.foodres.2015.02.017.
https://www.sciencedirect.com/science/ar...
), with some adaptations. Microparticles and free culture (1g) were transferred to 9ml of peptone water in test tubes. Contents were then subjected to thermal conditions of 72°C for 15 seconds and 63°C for 30 minutes, after which tubes were immediately cooled by immersion on ice for 10min. Finally, aliquots were collected and probiotic cultures were counted according to Section “Viable cell count”.

Assessment of the survival of encapsulated Lactobacillus acidophilus La-05 and Bifidobacterium Bb-12 exposed to simulated gastrointestinal conditions

The method proposed by MADUREIRA et al. (2011MADUREIRA, A. R. et al. Protective effect of whey cheese matrix on probiotic strains exposed to simulated gastrointestinal conditions. Food Research International, n. 44, p. 465-470, 2011. Available from: https://www.sciencedirect.com/science/article/pii/S0963996910003182>. Accessed: Nov. 30, 2017. doi: 10.1016/j.foodres.2010.09.010.
https://www.sciencedirect.com/science/ar...
), with some adaptations, was used to submit the microparticles to simulated gastrointestinal conditions. Viability of the bacteria was determined in media simulating the different sections of the gastrointestinal tract, such as esophagus/stomach (addition of pepsin, pH adjusted to 2.0 for 90min), duodenum (addition of pancreatin and bile salts, pH adjusted to 5.0 for 20min), and ileum (pH adjusted to 7.5 for 90min). Analysis was conducted on a TE 421 Shaker (Tecnal, Piracicaba, SP, Brazil) at a temperature of 37°C, simulating the temperature of the human body. Finally, aliquots were removed after 90min (esophagus or stomach), 110min (duodenum), or 200min (ileum) to determine the survival of free and microencapsulated Lactobacillus acidophilus La-5. Probiotic cultures were counted in MRS medium as described in Section “Viable cell count”.

Viability of microparticles during storage at different temperatures

Viability of the microencapsulated microorganisms was determined by enumeration in MRS agar, as described in Section 2.3. Microparticles were examined after storage for 0, 15, 30, 45, 60, 75, 90, 105, and 120 days at 25°C, −18°C and 7°C.

Statistical analysis

Data were submitted to analysis of variance (ANOVA) using Statistic version 7.0 software (2004; Statsoft Inc., Tulsa, OK, USA), followed by Tukey’s means comparison test at a level of 5% significance of treatments showing possible significant differences. All experiments were performed in triplicate; data are expressed as means ± standard deviations.

RESULTS AND DISCUSSION:

Viability, encapsulation efficiency, water activity and moisture of microparticles produced t different drying temperatures

The viability of the microparticles containing Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) produced at the different drying temperatures can be seen in table 1. The air inlet temperature of 140°C had a significant effect (P<0.05) in relation to the other conditions evaluated, presenting the lowest results for viability for both microparticles studied (ML and MB). Temperature of 130°C showed the greatest viability for Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles, but there was no significant difference (P<0.05) among temperatures of 110, 120 and 130°C. Similar results were reported by BUSTAMANTE et al. (2017BUSTAMANTE, M. et al. Effective Lactobacillus plantarum and Bifidubaterium infantis encapsulated with chia seed (Salvia Hispanica L.) and flax seed (Linun Usitatissimum L.) mucilage and soluble protein by spray drying. Food Chemistry, v. 1, n. 216, p. 97-105, 2017. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27596397>. Accessed: Nov. 11, 2017. doi: 10.1016/j.foodchem.2016.08.019.
https://www.ncbi.nlm.nih.gov/pubmed/2759...
) when encapsulating Lactobacillus acidophilus with mucilage extracted from the chia seed at two different spray dryer temperatures (110°C and 140°C). These authors reported lower survival of encapsulated Lactobacillus acidophilus at 140°C. ARSLAN et al. (2015ARSLAN, S. et al. Microencapsulation of probiotic Saccharomyces cerevisiae var. boulardii with diferente wall material by spray drying. LWT- Food Science and Technology, v. 63, p. 685-690, 2015. Available from: https://www.sciencedirect.com/science/article/pii/S0023643815001875>. Accessed: Nov. 10, 2017. doi: 10.1016/j.lwt.2015.03.034.
https://www.sciencedirect.com/science/ar...
) reported that an increase in the air inlet temperature of the spray dryer resulted in decreased viability and lower survival rates of Saccharomyces cerevisiae var. boulardi. Increase in the inlet temperature of the spray dryer consequently causes an increase in the outlet temperature. PISPAN et al. (2013PISPAN, S. et al. Comparison of cell survival rates of E. coli K12 and L. acidophilus undergoing spray drying. Food and Bioproducts Processing, v. 91, p. 362-369, 2013. Available from: http://www.sciencedirect.com/science/article/pii/S0960308513000060>. Accessed: Dec. 13, 2017. doi: 10.1016/j.fbp.2013.01.005.
http://www.sciencedirect.com/science/art...
) explained that an increase in the outlet temperature directly increases the temperature at which the microparticles are exposed. Conversely, a reduction in the outlet temperature results in a longer drying time. Thus, viability losses during the spray drying process can arise from dehydration and high temperatures. These two mechanisms occurring at the same time cause a negative effect on the survival of probiotic microorganisms (PEIGHAMBARDOUST et al., 2011PEIGHAMBARDOUST, S. H. et al. Application of spray drying for preservation of latic acid starter cultures: a review. Trends in Food Science & Technology, v. 22, p. 215-224, 2011. Available from: https://www.sciencedirect.com/science/article/pii/S0924224411000100>. Accessed: Nov. 19, 2017. doi: 10.1016/j.tifs.2011.01.009.
https://www.sciencedirect.com/science/ar...
; RIVEROS et al., 2009RIVEROS, B. et al. Spray drying of a vaginal probiotic strain of Lactobacillus. Drying Technology , v. 27, p. 123-132, 2009. Available from: http://www.tandfonline.com/doi/abs/10.1080/07373930802566002>. Accessed: Nov. 16, 2017. doi: 10.1080/07373930802566002.
http://www.tandfonline.com/doi/abs/10.10...
). The encapsulation efficiency (Table 1) ranged from 81.17 to 84.61% and 74.31 to 79.73% for the microparticles containing Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB), respectively. Therefore, it is possible to observe that Lactobacillus acidophilus La-5 presented greater resistance on the spray-drying conditions compared to Bifidobacterium Bb-12. RANADHEERA et al. (2015) evaluated viability of Bifidobacterium Bb-12, Lactobacillus acidophilus La-5 and Propionibacterium jansenii encapsulated in spray dryer reported greater loss viability for Bifidobacterium Bb-12. The current study showed greater resistance to the drying conditions used for the Lactobacillus acidophilus (LAC4) culture compared to Bifidobacterium lactis (B01). The highest encapsulation efficiency for both microparticles produced, ML (84.61%) and MB (79.73%) was observed at 130°C. FAVARO-TRINDADE AND GROSSO (2002)FAVARO-TRINDADE; GROSSO. Microencapsulation of L. acidophilus (La-05) and B. lactis (Bb-12) and evaluation of their survival at the pH values of the stomach and in bile. Journal of Microencapsulation, v. 19, n. 4, p. 485-494, 2002. Available from: https://www.ncbi.nlm.nih.gov/pubmed/12396385>. Accessed: Apr. 11, 2018. doi. 10.1080/02652040210140715.
https://www.ncbi.nlm.nih.gov/pubmed/1239...
and LIAN et al. (2002LIAN, W. C.; et al. Survival of bifidobacteria after spray drying. International Journal Food Microbiology, v. 74, p. 79-86, 2002. Available from: http://www.sciencedirect.com/science/article/pii/S0168160501007334>. Accessed: Dec. 14, 2017. doi: 10.1016/S0168-1605(01)00733-4.
http://www.sciencedirect.com/science/art...
) reported that different strains of microorganisms may vary in their ability to tolerate the high temperatures imposed during spray drying.

Table 1
Viability, encapsulation efficiency, water activity and moisture of microparticles containg Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) produced at different inlet temperatures in the spray dryer.

In the evaluation of the effect of different input temperatures on the water activity (Table 1) of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles, we observed that the temperature of 140°C had a significant influence (P<0.05), presenting the lowest water activity content for both studied microparticles. Nonetheless, the water activity reported in the different evaluated temperatures for the Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 microparticles ranged from 0.195 to 0.289. Thus, these results are as expected for microparticles dried by spray dryer (0.150 to 0.300) to ensure their microbiological stability (CORCORAN et al., 2004CORCORAN, B. M., et al. Comparative survival of probiotic lactobacilli spray dried in the presence of prebiotic substances. Journal of Applied Microbiology, v, 96, p. 1024-1039, 2004. Availabe from: < Availabe from: https://www.ncbi.nlm.nih.gov/pubmed/15078519 >. Accessed: Nov. 13, 2016. doi: 10.1111/j.1365-2672.2004.02219.x.
https://www.ncbi.nlm.nih.gov/pubmed/1507...
; ARSLAN et al., 2015ARSLAN, S. et al. Microencapsulation of probiotic Saccharomyces cerevisiae var. boulardii with diferente wall material by spray drying. LWT- Food Science and Technology, v. 63, p. 685-690, 2015. Available from: https://www.sciencedirect.com/science/article/pii/S0023643815001875>. Accessed: Nov. 10, 2017. doi: 10.1016/j.lwt.2015.03.034.
https://www.sciencedirect.com/science/ar...
).

The microparticles moisture content ranged from 4.60% to 5.71% (Table 1) and the lowest moisture contents were observed as the inlet temperature of the spray dryer was raised. FERRARI et al. (2012FERRARI, C. C., et al. Spray drying of blackberry pulp using maltodextrin as carrier agent. Brazilian Journal Food Technology, v. 15, p. 157-165, 2012. Available from: http://www.scielo.br/pdf/bjft/v15n2/aop_1411.pdf>. Accessed: Nov. 10, 2017. doi: 10.1590/S1981-67232012005000009.
http://www.scielo.br/pdf/bjft/v15n2/aop_...
) reported that higher temperatures imply a higher rate of heat transfer to the microparticles, resulting in a higher water evaporation and consequently, low moisture contents are obtained. Results obtained in the present research are in accordance with those reported by other authors who recommend that the moisture content should be around 4-5% to guarantee better storage stability (CHAVEZ & LEDEBOER, 2007CHAVEZ, B. E.; LEDEBOER, A. M. Drying of probiotics: optimixation of formulation and process to enchance storage survival. Drying Technology, v. 25, p. 1193-1201, 2007. Available from: http://www.tandfonline.com/doi/abs/10.1080/07373930701438576>. Accessed: Nov. 11, 2017. doi: 10.1080/07373930701438576.
http://www.tandfonline.com/doi/abs/10.10...
). In this sense, studies have shown that a lower inlet temperature and, consequently output, results in increased post-encapsulation viability; however, this condition may imply greater moisture and water activity, which adversely affects the prolonged storage of powders (PEIGHAMBARDOUST et al., 2011PEIGHAMBARDOUST, S. H. et al. Application of spray drying for preservation of latic acid starter cultures: a review. Trends in Food Science & Technology, v. 22, p. 215-224, 2011. Available from: https://www.sciencedirect.com/science/article/pii/S0924224411000100>. Accessed: Nov. 19, 2017. doi: 10.1016/j.tifs.2011.01.009.
https://www.sciencedirect.com/science/ar...
; VESTERLUND et al., 2012VESTERLUND, S. et al. Water activity in dry foods containing live probiotic bacteria should be carefully considered: A case study with Lactobacillus rhamnosus GG in flaxseed. International Journal of Food Microbiology, v. 157, p. 319-321, 2012. Available from: https://www.ncbi.nlm.nih.gov/pubmed/22663981>. Accessed: Dec. 22, 2017. doi: 10.1016/j.ijfoodmicro.2012.05.016.
https://www.ncbi.nlm.nih.gov/pubmed/2266...
). Thus, the relevance of the study of different drying temperatures, not only on the viability of the microorganisms, but also their influence on the physical characteristics of the microparticles is emphasized. MORGAN et al. (2006MORGAN, C. A. et al. Preservation of micro-organisms by drying: a review. Journal Microbiology Methods, 66, 183-193, 2006. Available from: https://www.ncbi.nlm.nih.gov/pubmed/16632005>. Accessed: Oct. 19, 2017. doi: 10.1016/j.mimet.2006.02.017.
https://www.ncbi.nlm.nih.gov/pubmed/1663...
) reported that spray-drying temperatures are of great importance for the viability of bacteria and need to be optimized individually for every new application. In this context, Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles produced at 130°C were chosen to be evaluated in this study.

Morphology and size of microparticles

Scanning electron microscopy of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles can be observed in figure 1. The microparticles produced presented a rounded shape containing concavities. The same was observed by FAVARO-TRINDADE & GROSSO (2002) FAVARO-TRINDADE; GROSSO. Microencapsulation of L. acidophilus (La-05) and B. lactis (Bb-12) and evaluation of their survival at the pH values of the stomach and in bile. Journal of Microencapsulation, v. 19, n. 4, p. 485-494, 2002. Available from: https://www.ncbi.nlm.nih.gov/pubmed/12396385>. Accessed: Apr. 11, 2018. doi. 10.1080/02652040210140715.
https://www.ncbi.nlm.nih.gov/pubmed/1239...
and FRITZEN-FREIRE et al. (2012FRITZEN-FREIRE, C. B. et al. Microencapsulation of bifidobacteria by spray drying in the presence of prebiotics. Food Research International, v. 45, p. 306-312, 2012. Available from: https://www.sciencedirect.com/science/article/pii/S0963996911005539>. Accessed: Nov. 18, 2017. doi: 10.1016/j.foodres.2011.09.020.
https://www.sciencedirect.com/science/ar...
). These authors reported that these concavities are typical of spray-dried products. Moreover, it is possible to observe that microparticles of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) presented high porosity (Figure 1a) and ruptures in their structure (Figure 1b). This fact may be related to the low solids concentration (12% m/v) used in the formulations. Similar results were shown by PINTO et al. (2015PINTO, S. S. et al. Influence of microencapsulation with sweet whey and prebiotic on the survival of Bifidobacterium BB-12 under simulated gastrointestinal conditions and heat treatments. LWT- Food Science and Technology , 1004-1009, 2015. Available from: http://www.sciencedirect.com/science/article/pii/S0023643815300372>. Accessed: Dec. 13, 2017. doi: 10.1016/j.lwt.2015.07.020.
http://www.sciencedirect.com/science/art...
) in the production of microparticles containing Bifidobacterium Bb-12 and using a concentration of 10% w/v in combinations with liquid whey, whey retentate, inulin and polydextrose.

Figure 1
Micrographs of the microparticles produced with 8g of gum arabic, 2g of maltodextrin, 1.9mL of glycerol, 0.1mL of tween 80 and Lactobacillus acidophilus La-5 (ML) e microparticles produced with 8g of gum arabic, 2g of maltodextrin, 1.9mL of glycerol, 0.1mL of tween 80 and Bifidobacterium Bb-12 (MB).

The particle size observed for the microparticles of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) was 4.85 and 8.75, respectively. RAJAM & ANANDHARAMAKRISHNAN (2015) reported particle sizes from 6.68 to 23.89µm for microparticles containing Lactobacillus plantarum (MTCC 5422) using oligofructose, whey protein isolate and denatured whey protein isolate at a concentration of 20% (w/v). ARSLAN et al. (2015ARSLAN, S. et al. Microencapsulation of probiotic Saccharomyces cerevisiae var. boulardii with diferente wall material by spray drying. LWT- Food Science and Technology, v. 63, p. 685-690, 2015. Available from: https://www.sciencedirect.com/science/article/pii/S0023643815001875>. Accessed: Nov. 10, 2017. doi: 10.1016/j.lwt.2015.03.034.
https://www.sciencedirect.com/science/ar...
) verified particle sizes that ranged between 8.56 and 21.38µm by encapsulating Saccharomyces cerevisiae var. boulardii using gelatin, gum Arabic, maltodextrin, modified starch, whey protein concentrate and pea protein isolate as encapsulating agents. Smaller particle sizes shown in the present research may be related to the different film-forming and gelling properties of the materials used in the microencapsulation process. In addition, according to KUROZAWA et al. (2009KUROZAWA, L. E. et al. Effect of maltodextrin and gum arabic on water sorption and glass transition temperature of spray dried chicken meat hydrolysate protein. Journal of Food Engineering , v. 91, p. 287-296, 2009. Available from: https://www.sciencedirect.com/science/article/pii/S0260877408004470>. Accessed: Dec. 05, 2017. doi: 10.1016/j.jfoodeng.2008.09.006.
https://www.sciencedirect.com/science/ar...
), higher concentrations of encapsulating agents in the feed solution promote an increase in particle size. However, it is worth mentioning that microparticles obtained by spray dryer presented a desirable size, once that smaller particles are preferred to ensure homogeneity and quality when applied to food (BURGAIN et al., 2011BURGAIN, J. et al. Encapsulation of probiotic living cells: from laboratory scale to industrial applications. Journal of Food Engineering, v. 104, p. 467-483, 2011. Available from: https://www.sciencedirect.com/science/article/pii/S026087741000631X>. Accessed: Nov. 15, 2017. doi: 10.1016/j.jfoodeng.2010.12.031.
https://www.sciencedirect.com/science/ar...
).

Resistance of microparticles to heat treatment

The microparticles of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) were evaluated to the heat treatments of 63°C/30min and 72°C/15s (Table 2). The microparticles of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) presented reductions of 1.91 and 1.93, at 63°C/30min and 1.36 and 1.42 at 72°C/15s, respectively. Thus, Lactobacillus acidophilus La-5 (ML) microparticles presented higher resistance to the thermal treatments studied. FAVARO-TRINDADE & GROSSO (2002) and LIAN et al. (2002LIAN, W. C.; et al. Survival of bifidobacteria after spray drying. International Journal Food Microbiology, v. 74, p. 79-86, 2002. Available from: http://www.sciencedirect.com/science/article/pii/S0168160501007334>. Accessed: Dec. 14, 2017. doi: 10.1016/S0168-1605(01)00733-4.
http://www.sciencedirect.com/science/art...
) have shown in previous studies that different strains of microorganisms can vary their ability to resist high temperatures. Bifidobacteria are known to be more susceptible to high temperatures than lactobacilus (DOLEYRES & LACROIX, 2005DOLEYRES, Y.; LACROIX, C. Technologies with free and immobilised cells for probiotic bifidobacteria production and protection. International Dairy Journal, v. 15, p. 973-988, 2005. Available from: https://www.sciencedirect.com/science/article/pii/S0958694605000063>. Accessed: Nov.05, 2017. doi: 10.1016/j.idairyj.2004.11.014.
https://www.sciencedirect.com/science/ar...
). However, it is noteworthy that for both thermal treatments studied, all viable cell counts of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles were superior than 6log CFU/g-1.

Table 2
Effect of heat treatments on the viability of microparticles containg Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) produced at inlet temperature of 130°C in the spray dryer.

Regarding the different applied thermal treatments, the higher temperature and the shorter time (72°C/15s) resulted in higher survival for the Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles. These results are in accordance with those reported by ZHANG, et al. (2015ZHANG, Y.; LIN, J. B.; ZHONG, Q. The increased viability of probiotic Lactobacillus salivarius NRRL B-30514 encapsulated in emulsions with multiple lipid-protein-pectin layers. Food Research International, v. 71, p. 9-15, 2015. Available from: https://www.sciencedirect.com/science/article/pii/S0963996915000782>. Accessed: Nov. 01, 2017. doi: 10.1016/j.foodres.2015.02.017.
https://www.sciencedirect.com/science/ar...
) and NUNES et al. (2017NUNES, G. L. et al. Inulin, hi-maize, and trehalose as thermal protectants for increasing viability of Lactobacillus acidophilus encapsulated by spray drying. LWT- Food Science and Technology , v. 89, p. 128-133, 2017. Available from: https://www.sciencedirect.com/science/article/pii/S0023643817307703>. Accessed: Dec. 12, 2017. doi: 10.1016/j.lwt.2017.10.032.
https://www.sciencedirect.com/science/ar...
), who reported better survival of Lactobacillus salivarius NRR B-30514 and Lactobacillus acidophilus La-5 encapsulated by the emulsion and spray drying methods, respectively, when subjected to heat treatment at 72°C/15s in relative to 63°C/30min.

Exposure of microparticles to simulated gastrointestinal conditions

Table 3 shows viable cell counts of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles exposed to simulated gastrointestinal conditions. After 90 min incubation in the presence of a pepsin solution and pH adjusted to 2.0 (simulated esophagus/stomach), there was significantly decreased (P<0.05) of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles compared to the initial count of 3.58 and 2.85log cycles, respectively. HOLKEN et al. (2016HOLKEN, A. T. et al. Development and characterization of alginate microcapsules containing Bifidubacterium BB12 produced by emulsification/internal gelation followed by freeze drying. LWT - Food Science and Technology, 71, 302-308, 2016. Available from: https://www.sciencedirect.com/science/article/pii/S0023643816301967>. Accessed: Dec. 11, 2017. doi: 10.1016/j.lwt.2016.04.012.
https://www.sciencedirect.com/science/ar...
) and NUNES et al. (2017NUNES, G. L. et al. Inulin, hi-maize, and trehalose as thermal protectants for increasing viability of Lactobacillus acidophilus encapsulated by spray drying. LWT- Food Science and Technology , v. 89, p. 128-133, 2017. Available from: https://www.sciencedirect.com/science/article/pii/S0023643817307703>. Accessed: Dec. 12, 2017. doi: 10.1016/j.lwt.2017.10.032.
https://www.sciencedirect.com/science/ar...
) verified a similar behaviour when encapsulating Bifidobacterium Bb-12 by emulsification/internal gelation and Lactobacillus acidophillus by spray drying, respectively. These authors explained that this reduction in log cycles does not necessarily imply a loss of viability, since the microparticles should not have ruptured at a pH value 2.0. Subsequently, when the microparticles are in contact with bile salts and pH 5.0 (section of the gastrointestinal tract comprising the duodenum), it was observed an increased number of viable cells (Table 4). Similar results were reported by HOLKEM et al. (2016), NUNES et al. (2017)GOMES, A. M. P.; MALCATA, F. X. Bifidobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trends in Food Science & Technology, v. 10, p. 139-157, 1999. Available from: https://www.sciencedirect.com/science/article/pii/S0924224499000333>. Accessed: Dec. 14, 2017. doi: 0.1016/S0924-2244(99)00033-3.
https://www.sciencedirect.com/science/ar...
and RAJAM et al. (2013RAJAM, R. P. et al. Effect of whey protein-alginate wall systems on survival of microencapsulated Lacotobacillus plantarum in simulated gastrointestinal conditions. Journal of Functional Foods, v. 4, p. 891-898, 2013. Available from: https://www.sciencedirect.com/science/article/pii/S1756464612001016>. Accessed: Oct. 15, 2017. doi: 10.1016/j.jff.2012.06.006.
https://www.sciencedirect.com/science/ar...
). The increase in viable cell count under these conditions probably resulted from a recovery of the sub-injured cells (PICOT & LACROIX, 2004). In the last section of the simulated gastrointestinal tract, the ileum (pH 7.5), the microparticles of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) continued to show a significant increase (P<0.05) in the number of viable cells (Table 3). As the pH was rising, the number of bacterial cells was increasing. The acid conditions of the stomach cause a dormant state in the bacterial cells, as the pH goes up they regain their growth (MOUMITA et al., 2017MOUMITA, S. et al. Evaluation of the viability of free and encapsulated lactic acid bacteria using in-vitro gastro intestinal model and survivability studies of symbiotic microcapsules in dry food matrix during storage. LWT- Food Science and Technology, v. 77, p. 40-477, 2017. Available from: http://www.sciencedirect.com/science/article/pii/S0023643816307666>. Accessed: Dec. 10, 2017. doi: 10.1016/j.lwt.2016.11.079.
http://www.sciencedirect.com/science/art...
). The microparticles of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) after exposure to simulated gastrointestinal tract conditions presented reductions of 1.72 and 2.04log cycles, respectively. Therefore, the Lactobacillus acidophilus La-5 (ML) microparticles were more resistant to simulated gastrointestinal conditions than Bifidobacterium Bb-12 (MB). These results differ from those reported by PEDROSO et al. (2012PEDROSO, D. L. et al. Protection of Bifidobacterium lactis and Lactobacillus acidophilus by microencapsulation using spray-chilling. International Dairy Journal, v. 26, p.127-132, 2012. Available from: https://www.sciencedirect.com/science/article/pii/S0958694612001021>. Accessed: Nov. 26, 2017. doi: 10.1016/j.idairyj.2012.04.008.
https://www.sciencedirect.com/science/ar...
) who reported greater gastrointestinal survival for Bifidobacterium lactis compared to Lactobacillus acidophilus. According to GOMES & MALCATA (1999GOMES, A. M. P.; MALCATA, F. X. Bifidobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trends in Food Science & Technology, v. 10, p. 139-157, 1999. Available from: https://www.sciencedirect.com/science/article/pii/S0924224499000333>. Accessed: Dec. 14, 2017. doi: 0.1016/S0924-2244(99)00033-3.
https://www.sciencedirect.com/science/ar...
) and KÕLL et al. (2008)KÕLL, P. et al. Characterization of oral lactobacillus as potential probiotics for oral health. Oral Microbiology Immunology, v. 23, p. 139-47. Available from: https://www.ncbi.nlm.nih.gov/pubmed/18279182>. Accessed: Nov. 10, 2017. doi: 10.1111/j.1399-302X.2007.00402.x.
https://www.ncbi.nlm.nih.gov/pubmed/1827...
, there is a variation in the ability of Bifidobacterium and Lactobacillus acidophilus to resist acid and bile conditions. These authors further reported that these properties are specific to strains and species.

Table 3
Viability of microparticles containing Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) produced at inlet temperature of 130°C in the spray dryer against simulated gastrointestinal.

Stability of microparticles during storage at different temperatures

Table 4 shows the viability of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles at room temperature (25°C), below freezing (-18°C) and under refrigeration (7°C). Room temperature (25°C) was the most damaging to the viability of Lactobacillus acidhopilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles, promoting reductions after 120 days’ storage between 3.82 and 3.51logs CFU/g-1. HUANG et al. (2017HUANG, S. et al. Spray Drying of probiotics and other food-grade bacteria: A review. Trendes in Food Science & Techonology, v. 63, p. 1-17, 2017. Available from: https://www.sciencedirect.com/science/article/pii/S0924224416303247>. Accessed: Dec. 18, 2017. doi: 10.1016/j.tifs.2017.02.007.
https://www.sciencedirect.com/science/ar...
) microencapsulated by spray drying Lactobacillus casei BL23 and Propionibacterium freudenreichii TG P20 and verified that storage at room temperature (25°C) resulted in greater viability loss. KOTULA (2008KOTULA, L. U. 2008. Application of a Propionibacterium jensenii 702 for human consumption. Australia: University of Newcastle, Ph.D., thesis.) reported that storage of probiotic powders above refrigeration temperatures increases rates of bacterial metabolism, which can lead to the accumulation of toxic residues and lead to a reduction in viability.

Table 4
Effect of room temperature (25°C), freezing (−18°C), and refrigeration (7°C) on the viability of microparticles containing Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) produced at inlet temperature of 130 °C in the spray dryer during storage for 120 days.

For freezing and refrigeration temperatures, microparticles presented losses of 2.81 and 2.22log cycles for Lactobacillus acidophilus La-5 (ML) and 2.42 and 2.12 for Bifidobacterium Bb-12 (MB), respectively. Thus, the refrigeration temperature promoted the greatest viability during storage for 120 days for both studied microparticles. OLIVEIRA et al. (2007OLIVEIRA, A. C. et al. Microencapsulation of B. lactis (BI 01) and L. acidophilus (LAC 4) by complex coacervation followed by spouted-bed drying. Drying Technology , v. 25, p. 1687-1693, 2007. Available from: http://www.tandfonline.com/doi/abs/10.1080/07373930701590939>. Accessed: Dec. 15, 2017. doi: 10.1080/07373930701590939.
http://www.tandfonline.com/doi/abs/10.10...
) showed that L. acidophilus exhibited greater viability at a storage temperature of 7°C, thus reporting similar results. Among microparticles of Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB), it is possible to observe that for the different evaluated temperatures, Bifidobacterium Bb-12 (MB) presented the smallest reductions during the 120 days of storage. However, considering the minimum level of 106logs CFU/g-1 (TALWALKAR et al., 2004TALWALKAR, A. et al. Effect of packaging materials and dissolved oxygen on the survival of probiotic bacteria in yoghurt. International Journal of Food Science and Technology, v. 39, p. 605-611, 2004. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2621.2004.00820.x/abstract>. Accessed: Oct. 12, 2017. doi. 10.1111/j.1365-2621.2004.00820.x.
http://onlinelibrary.wiley.com/doi/10.11...
), Bifidobacterium Bb-12 (MB) microparticles had a shelf life of only 60 days at 25°C and 105 days at -18°C while the Lactobacillus acidophilus La-5 (ML) microparticles remained viable throughout the storage period at all studied temperatures. Similar results were reported by PEDROSO et al. (2012PEDROSO, D. L. et al. Protection of Bifidobacterium lactis and Lactobacillus acidophilus by microencapsulation using spray-chilling. International Dairy Journal, v. 26, p.127-132, 2012. Available from: https://www.sciencedirect.com/science/article/pii/S0958694612001021>. Accessed: Nov. 26, 2017. doi: 10.1016/j.idairyj.2012.04.008.
https://www.sciencedirect.com/science/ar...
) who microencapsulated Bifidobacterium lactis and Lactobacillus acidophilus using spray-chilling. However, BUSTAMANTE et al. (2017BUSTAMANTE, M. et al. Effective Lactobacillus plantarum and Bifidubaterium infantis encapsulated with chia seed (Salvia Hispanica L.) and flax seed (Linun Usitatissimum L.) mucilage and soluble protein by spray drying. Food Chemistry, v. 1, n. 216, p. 97-105, 2017. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27596397>. Accessed: Nov. 11, 2017. doi: 10.1016/j.foodchem.2016.08.019.
https://www.ncbi.nlm.nih.gov/pubmed/2759...
) found greater viability for Bifidobacterium infantis in comparison to Lactobacillus plantarum incorporated in instant juice powder stored at 4°C for 45 days. According to MARTIN et al. (2015MARTIN, M. J. et al. Microencapsulation of bacteria: A review of different technologies and their impact on the probiotic effects. Innovative Food Science and Technologies, v. 27, p. 15-25, 2015. Available from: https://www.sciencedirect.com/science/article/pii/S146685641400160X>. Accessed: Nov. 24, 2017. doi: 10.1016/j.ifset.2014.09.010.
https://www.sciencedirect.com/science/ar...
) different probiotic strains present distinct abilities to resist environmental conditions such as oxygen, pH, light and temperature. In addition, the conditions of the microencapsulation process are of great importance for the microorganisms to remain viable during their storage (OLIVEIRA et al., 2007OLIVEIRA, A. C. et al. Microencapsulation of B. lactis (BI 01) and L. acidophilus (LAC 4) by complex coacervation followed by spouted-bed drying. Drying Technology , v. 25, p. 1687-1693, 2007. Available from: http://www.tandfonline.com/doi/abs/10.1080/07373930701590939>. Accessed: Dec. 15, 2017. doi: 10.1080/07373930701590939.
http://www.tandfonline.com/doi/abs/10.10...
).

CONCLUSION:

The inlet temperature of 130°C in the spray dryer promoted the highest viability and encapsulation efficiency for the Lactobacillus acidophilus La-5 (ML) and Bifidobacterium Bb-12 (MB) microparticles, submitted to different drying temperatures. The Lactobacillus acidophilus La-5 (ML) microparticles showed greater viability when exposed to thermal treatments and gastrointestinal simulation. In the storage viability for 120 days, the refrigeration temperature (7°C) was the one that maintained the highest viability for both produced microparticles. However, only the microparticles of Lactobacillus acidophilus La-5 (ML) maintained their counts higher than 6log CFU/g-1 at all temperatures that were studied (25, -18 and 7°C). Bifidobacterium Bb-12 (MB) microparticles had a 60 days shelf life at 25°C and 105 days at -18°C, thus demonstrating that they could be applied to food products with shorter shelf life. Among the studied microparticles, Lactobacillus acidophilus La-5 (ML) showed greater viability and resistance under the conditions evaluated in this research or study.

ACKNOWLEDGEMENTS

The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) by the scholarship granted to the first author and the availability of use of the laboratories in the Departments of Chemistry and Farmacy in Universidade Federal de Santa Maria.

REFERENCES:

  • 0
    CR-2018-0035.R1

Publication Dates

  • Publication in this collection
    2018

History

  • Received
    22 Jan 2018
  • Accepted
    18 Apr 2018
  • Reviewed
    18 May 2018
Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
E-mail: cienciarural@mail.ufsm.br