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LWT - Food Science and Technology

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Viability of Lactobacillus acidophilus NRRL B-4495 encapsulated with highmaize starch, maltodextrin, and gum arabic

Vondel Reyesa, Arranee Chotikoc, Alexander Chouljenkoa, Subramaniam Sathivela,b,∗

a School of Nutrition and Food Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA, 70803-4300, USAbDepartment of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, LA, 70803-4300, USAc Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi, Pathum Thani, 12110, Thailand

A R T I C L E I N F O

Keywords:ProbioticsL. acidophilusSpray dryingMaltodextrinGum arabic

A B S T R A C T

The effects of different protective agents, high maize starch (HM), maltodextrin (MD), and gum arabic (GA) onthe viability of Lactobacillus acidophilus after spray drying and during storage at different conditions were in-vestigated. Also, the physicochemical properties of the spray dried powders were evaluated. Lactobacillus acid-ophilus NRRL B-4495 (LA) was suspended in a 20% w/v solution of HM, MD, or GA. The solutions were sepa-rately spray dried at 140 °C to obtain LA powders: LAHM, LAMD, and LAGA. The powders were separately placedin aluminum bags and packed under 97 and 10% vacuum. The powders were stored at refrigerated (4 °C) and atroom (23 °C) temperatures for 60 days. The moisture content of LA powders ranged from 5.63 to 8.98% with theLAMD powders showing lower moisture content than LAGA and LAHM powders. More than 6 log CFU/g of LA/gpowder survived at 4 °C at 60 days of storage. LAGA and LAMD powders packed under 97% vacuum and storedat 4 °C had significantly higher cell viability than other powders. The study demonstrated that viability of LApowders packed under 97% vacuum and stored at refrigerated temperature meets the recommended levels tohave therapeutic effects.

1. Introduction

Probiotics are defined as “live microorganisms that, when ad-ministered in adequate amounts, confer a health benefit on the host”(FAO/WHO, 2001). Some of their benefits on human health includecontrol of irritable bowel syndrome, inflammatory bowel diseases,suppression of endogenous/exogenous pathogens, improving lactosetolerance, and reducing risk factors for colon cancer (Meng, Stanton,Fitzgerald, Daly, & Ross, 2008). These benefits are reached by differentmechanisms such as antimicrobial activity, immune, antimutagenic,and antigenotoxic effects, influence on enzyme activity, competitiveexclusion and colonization, among others (Nagpal et al., 2012;Papadimitriou et al., 2015). Lactobacilli species, such as L. acidophilus,L. rhamnosus, L. paracasei, and L. plantarum are the most commonprobiotic microorganisms (Saad, Delattre, Urdaci, Schmitter, &Bressollier, 2013). Due to the increasing demand for healthy, nutritious,and functional foods, the food industry is attempting to develop pro-biotic foods that can retain a high viability during both processing andstorage conditions (Soukoulis, Behboudi-Jobbehdar, Yonekura,Parmenter, & Fisk, 2014). However, probiotic viability in food productsis negatively affected by several factors, including the presence of

antimicrobial compounds, oxygen toxicity, post-acidification, and sto-rage temperature (Vasiljevic & Shah, 2008). Poor cell viability duringstorage has been reported in many probiotic foods as well as low sur-vival after consumption (Evivie, Huo, Igene, & Bian, 2017; Lin, Hwang,Chen, & Tsen, 2006; Martín, Lara-Villoslada, Ruiz, & Morales, 2015).

Encapsulation is one of the approaches to assure probiotic viability.This technology can help protect cell viability and functionality duringprocessing, storage, and delivery through the human gastrointestinaltract (de Vos, Faas, Spasojevic, & Sikkema, 2010). The cells are en-trapped within hydrocolloidal agents, resulting in a reduction of cellinjury and/or cell loss caused by adverse conditions (Iravani,Korbekandi, & Mirmohammadi, 2015). Spray drying is defined as aprocess in which a liquid feed is put in contact with a hot dryingmedium, leading to the evaporation of the liquid, obtaining a driedproduct in form of powders, granules, or agglomerates (Solval, 2011).This microencapsulation technique improves the survival of probioticsin food during processing and storage and also helps to protect theprobiotic cells against the harsh conditions in the gastrointestinal tract(Ranadheera, Evans, Adams, & Baines, 2015). Furthermore, this tech-nique allows the production of microencapsulated probiotic bacteriawith low production costs and higher productivity (Huang et al., 2017).

https://doi.org/10.1016/j.lwt.2018.06.017Received 7 March 2018; Received in revised form 29 May 2018; Accepted 6 June 2018

∗ Corresponding author. School of Nutrition and Food Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA, 70803-4300, USA.E-mail address: ssathivel@agcenter.lsu.edu (S. Sathivel).

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However, during this process cells are exposed to high temperature andosmotic stresses due to dehydration, which could result in the loss ofviability after spray drying and also during storage (Pérez-Chabela,Lara-Labastida, Rodriguez-Huezo, & Totosaus, 2013).

In order to protect probiotic cells against the harsh conditions theyare exposed during the drying process a variety of materials have beenevaluated including gum arabic (GA), alginate, gelatin, maltodextrin(MD), pectin, skimmed milk, resistant starch, chitosan, whey protein,among others (De Castro-Cislaghi, Carina Dos Reis, Fritzen-Freire,Lorenz, & Sant’Anna, 2012). GA is a natural composite of proteins andpolysaccharides and the most commonly used material in spray drying.Its structure contains a major group with highly branched poly-saccharide consisting of a galactose backbone with linked branches ofarabinose and rhamnose. The second group is a higher molecularweight arabinogalactan-protein complex (Arslan-Tontul & Erbas,2017). Maltodextrins are high molecular weight polysaccharides, pro-duced by starch hydrolysis. MD and other carbohydrates may con-tribute to increase the stability of spray-dried bacteria in terms of wateractivity, moisture content, pH, solubility, hygroscopicity, nutritionalcomposition, glass transition temperature, color, and fluidity (Sosaet al., 2016). Starches are another important component for micro-encapsulation that exhibit good film-forming qualities which may fur-ther protect encapsulated substances (Nunes et al., 2018). Resistantstarch is the small fraction of starch that resists hydrolysis by α-amylaseand pullulanase treatment in vitro and is not hydrolyzed to glucose inthe small intestine but is fermented in the colon (Raigond, Ezekiel, &Raigond, 2015). Due to these functional characteristics, high maizestarch was selected as the encapsulation matrix for the probiotic cells.According to Burgain, Gaiani, Linder, and Scher (2011), resistant starchcan be used as an encapsulating agent for targeted delivery of probioticcells in the human colon. Research regarding the effects of a singularencapsulating material on probiotic survival is common in the litera-ture. A comparison between some of the more widely used encapsulantsand their protective effects under different storage conditions is perti-nent to developing more effective probiotic delivery systems. The ob-jective of this investigation was to evaluate effects of different en-capsulating agents: high maize starch (HM), MD, and GA on theviability of L. acidophilus NRRL B-4495 during storage.

2. Materials and methods

2.1. Microorganism

L. acidophilus NRRL B-4495 (LA) was provided by ARS CultureCollection (Washington, DC). The frozen bacteria were activated twicein deMan Rogosa Sharpe (MRS) broth (Neogen Corporation, Lansing,MI). The strain (75mL) was inoculated in MRS broth (1500mL) andincubated at 37 °C for 16 h to reach stationary phase. The LA cell cul-tures were harvested and washed with sterile distilled water by cen-trifugation at 10000× g for 10min at 4 °C (Model J2-HC, BeckmanCoulter, Inc., CA).

2.2. Preparation of probiotics solutions

Three wall material solutions (200 g/L) were separately prepared.HM with approx. 56% resistant starch (High maize® 260, IngredionIncorporated, NJ), MD with a dextrose equivalent (DE) of 9–13, (NowFoods Company, Bloomingdale, IL), and GA (Frontier Co-op, Norway,IA) were dissolved in distilled water and autoclaved at 121 °C for15min. The wall material solutions were then mixed with LA cell cul-tures (∼109 CFU/mL) to produce LA solutions.

2.3. Spray drying of probiotic solutions

LA probiotic solutions mixed with the different wall materials wereseparately fed into a pilot-scale spray dryer (FT80/81 Tall Form Spray

Dryer Armfield Inc., Ringwood, UK) under co-current drying conditionsat the Food Processing Pilot Plant, Louisiana State UniversityAgricultural Center. The inlet ambient air was electrically heated by aresistance heater to a constant temperature of 140 °C. A feeding pumpdelivered the probiotic solutions through a two-fluid stainless steel typespray nozzle where they were atomized with an atomizing air pressureof 14.5 psig and sprayed into the main dryer chamber. The three spraydried probiotic powders produced (LAHM, LAMD, and LAGA) werecollected from the base of the cyclone vessel. Samples of approximately1.5 g of the spray dried powders were separately placed in 4″ x 6”aluminum bags and packed under 97% and 10% vacuum (Koch UV-550, Kansas City, MO). The packed powders were stored separately atrefrigerated (4 °C) and at room (23 °C) temperatures for up to 60 days inorder to analyze the cell viability during storage. Temperatures wereselected based on our preliminary studies.

2.4. Physicochemical properties of the probiotic powders

The probiotic powders were analyzed for water activity (aw),moisture content, and color. The water activity was measured using anAquaLab Pawkit (Decagon Devices, Inc., Pullman, WA). The moisturecontent was determined using a microwave-type moisture analyzer(Model 907875, CEM Corporation, Inc., Matthews, NC). The color ofprobiotic powders was measured using a chroma meter LabScan XE(Hunterlab, VA) equipped with a pulsed xenon lamp and a 13mmaperture diameter. CIELAB color scales L*, a*, and b* were used toexpress the results. The L* values measure the degree of lightness todarkness, a* values measure the degree of redness to greenness, and b*values assess the degree of yellowness to blueness.

2.5. Viability of probiotics before and after spray drying and during storage

Probiotic solutions mixed with the different wall materials andpowders were determined for cell viability by separately suspendingand homogenizing with a vortex mixer 1 g in 9mL of 0.85 g/100mLsterile saline solution. Serial dilutions were prepared from the initialsuspension in the sterile saline solution. The pour plating method usingMRS agar (Neogen Corporation, Lansing, MI) with 0.6 g CaCO3/100mL(Sigma-Aldrich, St. Louis, MO) was performed in triplicate. The plateswere incubated at 37 °C, enumerated after 48 h, and results expressed ascolony forming units per gram sample (CFU/g).

2.6. Scanning electron microscopy

The morphology of probiotic powders was observed using a scan-ning electron microscope (SEM, JSM-6610LV, JEOL Ltd. Japan).Samples were mounted on aluminum SEM stubs and then coated withplatinum in an Edwards S150 sputter coater (Edwards High VacuumInternational, Wilmington, MA) for 4min prior to observation at both1000× and 3000×magnification.

2.7. Statistical analysis

The data were analyzed using SAS (Statistical Analysis System)software version 9.4 (SAS Institute Inc., Cary, NC). Experiments wereperformed in triplicate and the data were reported as means ±standard deviation. Tukey's test at an alpha of 0.05 was carried out todetermine significant differences among the treatments.

3. Results and discussion

3.1. Physicochemical properties of the probiotic powders

Water activity (aw) of LA powders was not affected by type of wallmaterial. As shown in Table 1, aw values of LA powders ranged from0.26 to 0.35 and were not significantly different. The aw indicates free

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water (water not bound to molecules) which allows biochemical reac-tions to proceed. The aw of probiotic powders or products has an impacton maintaining cell viability. According to Viernstein, Raffalt, andPolheim (2005), lower aw (0.2–0.3) caused better cell viability duringstorage. However, membrane lipids could be oxidized, leading to via-bility reduction, if aw is lower than 0.1.

Table 1 shows the moisture content in wet basis of LA powderswhich varied between 5.63 and 8.98%. The MD (LAMD=5.63%)powders had lower moisture content than GA (LAGA=8.94%) and HM(LAHM=8.98%) treatments. The results were similar with those ofTonon, Brabet, Pallet, Brat, and Hubinger (2009) who reported thatspray-dried açai powders produced with MD (10 DE) had a lowermoisture content than those produced with GA. Encapsulating agentsplay an important role in moisture content of powders after spraydrying, which is related to their glass transition temperature (Tg). Tg isspecific to each amorphous material and is affected by molecularweight, chemical structure, and moisture content of the material. deBarros Fernandes, Borges, and Botrel (2014) defined Tg as the tem-perature at which a state of polymeric material changes from a glassyamorphous state to a rubbery state. Tg has an influence on duration ofparticle crust formation during the drying process. Shamaei, Seiiedlou,Aghbashlo, Tsotsas, and Kharaghani (2017) claim that during the crustformation the resistance for water vapor diffusion increases whichproduces microcapsules with higher moisture content. Droplets con-taining wall materials with lower Tg form the crust before the dropletscontaining materials with higher Tg (Pourashouri et al., 2014).Kurozawa, Park, and Hubinger (2009) reported that spray dried chickenbreast protein hydrolysate powders with 20% (w/w) MD(9≤DE≤ 12) which had a similar DE to the one used in this study hada higher Tg value compared to those produced with 20% (w/w) GA.Meanwhile, Freire, Fertig, Podczeck, Veiga, and Sousa (2009) report noTg detection for Hylon V, a high amylose (56%) maize starch, claimingthat the glass transition temperature can only be identified in these typeof samples with a moisture content above 13%. Therefore, it is possiblethat crust formation of droplets containing MD occurred later than thatof droplets with GA and HM. As a result, LAMD powders had lowermoisture content than LAHM and LAGA powders.

The color values of probiotic powders with different wall materialsare reported in Table 2. The color of LA powders is attributed to thecarrier color (da Silva et al., 2013). Powders with MD and HM had

significantly higher lightness (L*) than GA. This was because the colorsof MD and HM were white, while GA used in this experiment was lightbrown. The color of spray dried powders can be affected by the con-centrations (Comunian et al., 2011) and types (Fritzen-Freire et al.,2012) of wall materials. Color is an important quality indicator of thepowders produced during the spray drying process.

3.2. Scanning electron microscopy of spray-dried probiotic powders

The spray-dried LA powders were observed by SEM (Fig. 1). Spray-dried powders had a surface without mechanical fissures and the pre-sence of concavities. These concavities were the result of the rapidevaporation of the atomized liquid drops during spray drying (Fritzen-Freire et al., 2012). LAMD powders consisted of particles with a wrin-kled surface or with concavities. Rodríguez-Huezo et al. (2007) re-ported that the use of moderate inlet drying temperatures in spray-driedpowders (140 °C) could produce concavities, making the particlesstronger against mechanical fracture and solute diffusion. LAGA pow-ders particles showed an overall spherical shape with both smoothnessand shrinkage on their surface, which was also found in other studiesusing GA as a wall material for the production of spray-dried probioticpowders (Guergoletto, Busanello, & Garcia, 2017; Kingwatee et al.,2015).

3.3. Effect of wall materials on the viability of spray-dried L. acidophilusNRRL B-4495 powders

The viability during storage of LA powders with different wallmaterials is illustrated in Fig. 2. Initial cell counts in LAHM, LAMD, andLAGA probiotic solutions before spray drying were 9.06, 9.05, and 9.24log CFU/g, respectively. The number of viable cells of LA powdersdecreased by less than 1.25 log CFU/g after spray drying. After spraydrying, the number of viable cells in LAGA, LAHM, and LAMD powderswas 8.10, 7.90, and 7.82 log CFU/g, respectively. These results showedthat GA was the best protective agent of cells during the spray dryingprocess. Regarding the storage temperatures, the results showed thatthe cell viability of LA powders was more stable at refrigerated tem-perature (4 °C) than those stored at ambient temperature (23 °C). Theseresults are in agreement with other studies that evaluate the effect oftemperature on the viability of L. acidophilus during storage (Nuneset al., 2018; Ranadheera et al., 2015; Soukoulis et al., 2014). Accordingto Chávez and Ledeboer (2007) and Santivarangkna, Kulozik and Foerst(2007), the survival of probiotic bacteria is inversely related to thetemperature during storage conditions. Furthermore, De Castro-Cislaghi et al. (2012) state that the encapsulating agent also has a directeffect on the stability of the microencapsulated cells. Regarding thevacuum conditions, LA showed a higher survival at 97% vacuum thanat 10% vacuum when they were kept at 4 °C and at 23 °C. This suggeststhat lower levels of oxygen improved cell viability during storage(Chávez & Ledeboer, 2007). According to Champagne, Gardner, andRoy (2005), oxygen affects probiotic cells due to the intracellular pro-duction of hydrogen peroxide. In addition, Tripathi and Giri (2014)revealed that the production of free radicals from the oxidation ofcellular fats can be toxic to probiotic cells.

3.3.1. Viability of spray-dried L. acidophilus NRRL B-4495 microcapsulesstored at refrigerated (4 °C) temperature

GA showed a better protective effect on preserving LA during sto-rage at 4 °C (Fig. 2) than HM and MD. At the 60th day of storage, LAGA-vac powders had higher cell viability than the other powder sampleswith 7.36 log CFU/g of cells, corresponding to a 0.75 log unit reductionin viability, while the number of live bacteria for LAGA-air powders was6.98 log CFU/g. The number of live bacteria for LAMD-vac and LAMD-air powders at 60 days decreased to 7.18 and 6.66 log CFU/g, respec-tively. Meanwhile, the number of live bacteria for LAHM-vac powderswas 6.35 log CFU/g. The results showed that more than 6 log CFU/g of

Table 1Water activity and moisture content of probiotic powders.

Wall Material Water activity Moisture content(wet basis g/100 g)

HM 0.35 ± 0.01A 8.98 ± 0.50A

MD 0.26 ± 0.00A 5.63 ± 0.02A

GA 0.31 ± 0.05A 8.94 ± 1.62A

Values are means ± SD of triplicate determinations. AMeans ± SD with thesame letter in a column are not significantly different (P > 0.05). LA= L.acidophilus NRRL B-4495, HM=high maize starch, MD=maltodextrin, andGA=gum arabic.

Table 2Color values of probiotic powders.

HM MD GA

L* 91.02 ± 0.96A 93.67 ± 1.09A 87.03 ± 0.32B

a* −0.04 ± 0.01A −0.39 ± 0.01B 0.11 ± 0.11A

b* 5.55 ± 0.29A 2.47 ± 0.63B 3.16 ± 0.71AB

L*, a*, and b* are the degree of lightness to darkness, redness to greenness, andyellowness to blueness, respectively. Values are means ± SD of triplicate de-terminations. A,BMeans ± SD with the same letter in a row are not significantlydifferent (P > 0.05). LA= L. acidophilus NRRL B-4495, HM=high maizestarch, MD=maltodextrin, and GA=gum arabic.

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L. acidophilus survived at 4 °C at 60 days of storage and that the con-centration of the wall materials used was able to protect the en-capsulated cells. This is in agreement with the results reported by otherstudies that evaluated L. acidophilus viability using different en-capsulating agents during refrigerated storage. Their results showedthat encapsulation by spray drying and storage at refrigerated tem-peratures were able to improve the number of viable cells (Behboudi-Jobbehdar, Soukoulis, Yonekura, & Fisk, 2013; Maciel, Chaves, Grosso,& Gigante, 2014; Pispan, Hewitt, & Stapley, 2013; Riveros, Ferrer, &Borquez, 2009). Zhao, Sun, Torley, Wang, and Niu (2008) claim that aminimum concentration of L. acidophilus equivalent to 106 CFU/mL orgram of product is needed to have therapeutic benefits in the humanbody. Functional foods such as yogurt containing above 6 log CFU/g ormL of viable probiotic L. acidophilus at the time of consumption help topromote health benefits to the consumers (Machado et al., 2017).

3.3.2. Viability of spray-dried L. acidophilus NRRL B-4495 microcapsulesstored at room (23 °C) temperature

The L. acidophilus powders stored at room temperature (Fig. 3) ex-perienced a steep decline in cell viability during storage. At the 10thday of storage, the number of viable cells in all LA powders was aboveof 106 CFU/g. However, at the 20th and 30th day of storage, onlyLAMD-vac and LAGA-vac kept a cell viability of 106 CFU/g. There wereno viable cells of LA detected at 60 days of storage at room temperatureregardless of wall material and vacuum conditions, except for theLAMA-air powders which had low concentration LA (3.56 log CFU/g).Physical and chemical characteristics of MD might have contributed toincrease the stability of spray-dried LA during the storage more than theother wall materials did. Cell counts for all the LA powders stored atroom temperature failed to meet the guidelines for probiotic products(at least 6 log CFU/g). The results indicated that LA was highly sus-ceptible to the tested storage conditions. Soukoulis et al. (2014) statethat L. acidophilus is a thermo-sensitive probiotic strain and their

Fig. 1. Scanning electron micrographs of spray-dried L. acidophilus NRRL B-4495 powders. HM=high maize starch, MD=maltodextrin, GA= gum arabic, andLA= L. acidophilus NRRL B-4495. SD-LAHM= spray-dried microcapsules of LA with high maize starch, SD-LAMD= spray-dried microcapsules of LA with mal-todextrin, and SD-LAGA= spray-dried microcapsules of LA with gum arabic. Magnification: left side= 1000× , right side= 3000× .

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viability is critically affected by the operating spray drying conditions.Also, Fu and Chen (2011) classify L. acidophilus as a heat sensitivebacteria. According to Soukoulis et al. (2014), some lactobacilli strainshave greater thermotolerance throughout spray drying and this char-acteristic is strictly strain specific. It is likely that the effects of spraydrying on cell damage could subsequently affect the viability of L.acidophilus during the storage conditions.

4. Conclusion

The present study investigated the effect of three wall materials onthe viability of L. acidophilus NRRL B-4495 after spray drying andduring storage at different conditions and the physicochemical prop-erties of the spray dried powders. The LAMD powders had lowermoisture content and water activity than the LAGA and LAHM powders.L. acidophilus powders with GA and MD packed under 97% vacuum and

Fig. 2. Effect of different wall materials on the survival of spray-dried L. acidophilus NRRL B-4495 microcapsules stored under 97 and 10% vacuum at refrigerated(4 °C) temperature. A,BMeans ± SD with same letters between treatments at the same storage times are not significantly different (P > 0.05). a–eMeans ± SD withsame letters within treatments at different storage times are not significantly different (P > 0.05). HM=high maize starch, MD=maltodextrin, GA=gum arabic,Vac= 97% vacuum, Air= 10% vacuum, and LA= L. acidophilus NRRL B-4495. GA-Vac ( ), and GA-Air ( )= spray-dried LA with GA stored under 97 and 10%vacuum, respectively; MD-Vac ( ) and MD-Air ( )= spray-dried LA with HM stored under 97 and 10% vacuum, respectively; and HM-Vac ( ) and HM-Air ()= spray-dried LA with MD stored under 97 and 10% vacuum, respectively.

Fig. 3. Effect of different wall materials on the survival of spray-dried L. acidophilus NRRL B-4495 microcapsules stored under 97 and 10% vacuum at refrigerated(23 °C) temperature. A,BMeans ± SD with same letters between treatments at the same storage times are not significantly different (P > 0.05). a–fMeans ± SD withsame letters within treatments at different storage times are not significantly different (P > 0.05). HM=high maize starch, MD=maltodextrin, GA=gum arabic,Vac= 97% vacuum, Air= 10% vacuum, and LA= L. acidophilus NRRL B-4495. GA-Vac ( ), and GA-Air ( )= spray-dried LA with GA stored under 97 and 10%vacuum, respectively; MD-Vac ( ) and MD-Air ( )= spray-dried LA with HM stored under 97 and 10% vacuum, respectively; and HM-Vac ( ) and HM-Air ()= spray-dried LA with MD stored under 97 and 10% vacuum, respectively.

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stored at 4 °C had significantly higher cell viability than the otherpowder samples. The data obtained showed that more than 6 log CFU/gof L. acidophilus survived at 4 °C at 60 days of storage which meets therecommended levels to have therapeutic effects in the human host. Thetechnologies used to produce and store encapsulated LA are commonlyused in food applications, and are scalable to an industrial level. Theobtained results may be used to optimize combined processing andstorage conditions for survival of L. acidophilus NRRL B-4495 in morecomplex food systems.

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