Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Development of alginate-pectin microparticles with dairy whey usingvibration technology: Effects of matrix composition on the protection ofLactobacillus spp. from adverse conditions

Camila Eckerta,b, Wendell Dall Agnola,b, Danieli Dalléa,b, Vanessa Garcia Serpaa,b,Mônica Jachetti Maciela,c, Daniel Neutzling Lehna, Claucia Fernanda Volken de Souzaa,b,c,⁎

a Laboratory of Food Biotechnology, University of Vale do Taquari - Univates, Lajeado, RS, Brazilb Postgraduate Program in Biotechnology, University of Vale do Taquari - Univates, Lajeado, RS, Brazilc Postgraduate Program in Sustainable Environmental Systems, University of Vale do Taquari - Univates, Lajeado, RS, Brazil.

A R T I C L E I N F O

Keywords:EncapsulationLactic acid bacteriaCheese wheyWhey permeate

A B S T R A C T

In this study, lactic acid bacteria with probiotic potential, including Lactobacillus plantarum ATCC8014, L.paracasei ML33 and L. pentosus ML82, were encapsulated with whey-alginate-pectin (WAP) or whey permeate-alginate-pectin (PAP) by an extrusion process using vibrational technology, with the resulting microparticlesassessed for their resistance to adverse conditions. The aim was to assess the effect of the encapsulation wallmaterials on the viability of microorganisms, the encapsulation, refrigerated storage and simulated gastro-intestinal tract conditions, the kinetic parameters of acidification, and the morphology of microparticles. Thebacteria encapsulated with the WAP wall material were adequately protected. Furthermore, after three monthsof storage at 4 °C, the encapsulated bacteria exhibited a cell viability of> 6 log CFUmL−1. In addition, theencapsulated L. plantarum ATCC8014 and L. pentosus ML82 isolates exhibited the highest viability at the end ofthe storage period among the assayed isolates. Encapsulated bacteria showed greater resistance to acidic con-ditions than unencapsulated bacteria when exposed to simulated gastrointestinal tract conditions. The maximumrate of milk acidification by encapsulated Lactobacillus spp. was approximately three-fold lower than that ob-served for unencapsulated bacteria. The resulting size of the microparticles generated using both combinationsof wall materials used was approximately 150 μm. The cheese whey and whey permeate combined with alginateand pectin to adequately encapsulate and protect Lactobacillus spp. from the adverse conditions of the simulatedgastrointestinal tract and from refrigeration storage temperatures. Furthermore, the sizes of the obtained mi-croparticles indicated that the encapsulated materials are suitable for being incorporated into foods withoutchanging their sensory properties.

1. Introduction

Lactobacillus is a genus of Gram-positive, aerotolerant or anaerobicand strictly fermentative bacteria that produce lactic acid (homo-fermentative) or equimolar amounts of lactic acid and other compounds(heterofermentative), which play a key role in the production of fer-mented foods (Soccol et al., 2010). Furthermore, they are distributedthroughout the gastrointestinal tract (GIT) and are important health-promoting members of the endogenous microbiota of humans and otheranimals (Anal & Singh, 2007; Jay, 2000). Specifically, by associatingwith intestinal epithelium cells and modulating the immune system,they promote therapeutic effects related to disease prevention (Burgain,Gaiani, Linder, & Scher, 2011; Vanderpool, Yan, & Polk, 2008).

Commercial strains of Lactobacillus, such as L. rhamnosus GG®, L.casei Shirota® and L. acidophilus La5®, are used to prepare fermentedmilk products, which are sold worldwide without taking into accountthe physiological characteristics, health status and geographical loca-tion of the populations that consume them. However, autochthonous orendogenous strains that have potential as starter, nonstarter and/orprobiotic lactic cultures have been used to make fermented productswith improved health effects for consumers. This is because the dietaryhabits of a population directly affect the GIT microbiota, and the en-dogenous strains become more easily adaptable both to this ecosystemand to the supplemented foods (Santos et al., 2016; Sybesma, Kort, &Lee, 2015).

However, a number of Lactobacillus species exhibit low resistance in

https://doi.org/10.1016/j.foodres.2018.07.001Received 15 January 2018; Received in revised form 3 June 2018; Accepted 1 July 2018

⁎ Corresponding author at: Av. Avelino Tallini, 171, ZC 95914-014, Lajeado, RS, Brazil.E-mail address: claucia@univates.br (C.F. Volken de Souza).

Food Research International 113 (2018) 65–73

Available online 02 July 20180963-9969/ © 2018 Elsevier Ltd. All rights reserved.

T

industrial processes, in foods and in the GIT, requiring a protectivebarrier to ensure their effectiveness (Martín, Lara-Villoslada, Ruiz, &Morales, 2015). Encapsulation techniques are considered the mostsuitable for this purpose (Vivek, 2013), and this process is defined asthe technology of packaging structures into small, sealed capsules,which controllably release their contents under specific conditions(Burgain et al., 2011; De Vos, Faas, Spasojevic, & Sikkema, 2010; Desai& Jin Park, 2005).

Extrusion with vibrational technology, also known as vibrating-jetor prilling, is a method that enables the sterile and standardized pro-duction of capsules without their exposure to adverse conditions, suchas high or freezing temperatures, which may affect the viability of theencapsulated microorganism. In this encapsulation process, a liquidlaminar jet is ruptured by an overlapping vibration frequency. When afluid is extruded through a nozzle at certain flow rates, there is for-mation of a laminar jet that may break-up at different lengths as a resultof the stresses applied to it. These segments produce spherical andstandard droplets due to the surface tension, that is, they form smalldroplets that fall into a gelification solution promoting the formation ofthe microparticles. The production of spherical and standard capsules isa critical factor because it influences their physical and chemicalproperties, such as the stability and controlled release of the protectedsensitive material, and it is the main factor affecting the viability ofencapsulated microorganisms (Coghetto, Brinques, & Ayub, 2016; El-Salam & El-Shibiny, 2015; Shi et al., 2013; Whelehan & Marison, 2011).Several studies using encapsulation by vibrational technology haveindicated its applicability to bacterial cells and the need for improve-ments to encapsulation conditions, including the production of micro-capsules with standardized shapes and decreased sizes with respect toother methods, as well as protection from the GIT (Burgain et al., 2011;De Prisco, Maresca, Ongeng, & Mauriello, 2015; Del Gaudio, Colombo,Colombo, Russo, & Sonvico, 2005; Maresca et al., 2016; Martoni,Bhathena, Urbanska, & Prakash, 2008; Mazzitelli et al., 2008).

However, this method requires a gelling agent that promotes thesolidification of the microcapsule. Alginate (D-mannuronic acid and L-guluronic acid) is the most used biopolymer for this purpose due to itsfavorable characteristics, including its low cost and safe use in foods.Furthermore, numerous studies have shown the positive effect of algi-nate on the viability of probiotics under adverse conditions (Bekhit,Sánchez-González, Messaoud, & Desobry, 2016; Coghetto, Brinques, &Ayub, 2016; Pankasemsuk, Apichartsrangkoon, Worametrachanon, &Techarang, 2016; Sandoval-Castilla, Lobato-Calleros, García-Galindo,Alvarez-Ramírez, & Vernon-Carter, 2010; Su, Lin, & Chen, 2007; Voo,Ravindra, Tey, & Chan, 2011; Zaeim, Sarabi-Jamab, Ghorani,Kadkhodaee, & Tromp, 2017). However, microparticles made with al-ginate are very porous, causing rapid matrix diffusion and reducing thebarrier against unfavorable conditions (Martín et al., 2015). An alter-native is the use of alginate in combination with other gelling agents toimprove the resistance of the resulting microcapsule. Studies suggestthat pectins, commonly used to increase the viscosity and gel strengthof food products, may be added to the alginate solution used for en-capsulation to reinforce the gel and increase its protective effect.Moreover, pectins also contribute to dietary fiber content, increasingthe nutritional value of the microparticles (Belščak-Cvitanović et al.,2015; Naqash, Masoodi, Rather, Wani, & Gani, 2017; Sandoval-Castillaet al., 2010; Zhang, Lin, & Zhong, 2016).

Furthermore, due to their amphiphilic structure, milk proteins,particularly casein products and whey protein, have excellent surfaceproperties that promote adsorption at interfaces and help stabilizeemulsions. Those proteins are widely used as emulsifying agents, and incombination with other polymers, they contribute to the surface sta-bility of the microcapsule due to their structure, flexibility and ag-gregation state (El-Salam & El-Shibiny, 2015; Keogh & o'Kennedy,1999; Livney, 2010). Several approaches using milk proteins for en-capsulation have been reported in the literature (Arslan, Erbas, Tontul,& Topuz, 2015; Kent & Doherty, 2014; Khem, Small, & May, 2016; Shi

et al., 2013).Alternative and low-cost sources of these constituents are by-pro-

ducts of the dairy industry. The use of these by-products expands theiruses and minimizes waste, since the high organic load and large volumegenerated make them potential pollutants if discarded incorrectly(Carvalho, Prazeres, & Rivas, 2013; Krunić, Bulatović, Obradović,Vukašinović-Sekulić, & Rakin, 2015; Ribeiro et al., 2014). Cheese wheyis the main by-product of cheese and casein production, composed ofapproximately 50% milk total solids that contain approximately 100%milk lactose and 20% milk proteins (Smithers, 2008; Smithers, 2015).Whey permeate is another by-product with potential to recover pri-marily proteins, which are used in the development of value-addedproducts. Whey permeate is produced from the cheese whey ultra-filtration process and primarily consists of lactose and salts. Due to itshigh lactose content, whey permeate has stood out as a substrate for thedevelopment of probiotic microorganisms (Hugo, Bruno, & Golowczyc,2016; Mollea, Marmo, & Bosco, 2013).

In this context, and considering that to the best of our knowledge,no studies have used dairy by-products in wall materials to encapsulatemicroorganisms by extrusion with vibrational technology, the objectiveof this study was to use this method to encapsulate endogenous lacticacid bacteria from southern Brazil, including L. paracasei ML33 and L.pentosus ML82 and L. plantarum ATCC8014. Combinations of dairy by-products, cheese whey or whey permeate, with sodium alginate andpectin, were used as wall materials, and the encapsulated lactic acidbacteria were evaluated for their viability after being stored at re-frigeration temperature, simulated gastrointestinal tract resistance andmilk acidification potential. The three microorganisms were also eval-uated regarding the feasibility of the encapsulation process, and theprepared microparticles were characterized for their morphology byoptical and scanning electron microscopy.

2. Materials and methods

2.1. Materials

All chemical reagents and culture media were purchased fromMerck (KGaA, Darmstadt, Germany) or Sigma-Aldrich Co., Ltd. (St.Louis, MO, USA). Cheese whey and whey permeate, derived from theby-products of mozzarella cheese production, in both fresh and liquidform, were donated from dairy factories in the region and maintained at4 °C. L. plantarum ATCC8014 was obtained from the American TypeCulture Collection (ATCC). L. paracasei ML33 and L. pentosus ML82were isolated from bovine milk from the Taquari Valley region, RioGrande do Sul state, Brazil. Stocks of the Lactobacillus strains weremaintained at −80 °C in 25% (v/v) glycerol. Prior to the preparation ofbacterial suspensions for encapsulation, lactic acid bacteria were re-covered from the stock and maintained at 4 °C on Petri dishes con-taining Man, Rogosa and Sharpe (MRS) agar.

2.2. Microparticle production by extrusion with vibrational technology

To prepare a bacterial suspension for encapsulation, an isolatedLactobacillus colony was transferred to a vial containing MRS broth andincubated at 37 °C for 48 h. Next, the number of bacteria in the cellsuspension was standardized to approximately 109 CFUmL−1.Subsequently, the cells were centrifuged (2.370×g, 10min, 4 °C), wa-shed twice with phosphate buffer (10mM, pH 7.0) and resuspended ineach of the feed solutions.

Cheese whey or whey permeate combined with sodium alginate andpectin were used as wall materials to make microparticles. Dairy by-products were heat treated at 65 °C for 30min, while alginate andpectin were autoclaved (121 °C for 15min). The feed solutions wereprepared by mixing 1:1:1 cheese whey (CW) or whey permeate (WP),1.5% sodium alginate (w/v) and 1.25% pectin (w/v). The encapsulatedbacterial cells were prepared using an Encapsulator B-395 Pro (BÜCHI

C. Eckert et al. Food Research International 113 (2018) 65–73

66

Labortechnik AG, Meierseggstrasse 40, CH-9320 Flawil 1, Switzerland),equipped with an 80-μm nozzle and a syringe pump loaded with 15mLof feed solution containing the microbial cells, according to De Priscoet al. (2015). The instrument was operated at a 5mL/min feed rate,1740 Hz vibration frequency and 950mV electrode voltage. The mi-croparticles were solidified in 200mL of a 0.5M CaCl2 solution understirring for 30min. Finally, the suspensions containing the micro-particles were collected in sterile flasks, washed and filtered with ul-trapure water, resuspended in 100mL of phosphate buffer (10mM,pH 7.0) and stored at 4 °C. Microparticles made with cheese whey-al-ginate-pectin (M.WAP) and whey permeate-alginate-pectin (M.PAP)were produced for each Lactobacillus isolate (L. plantarum ATCC8014, L.paracasei ML33 and L. pentosus ML82). The encapsulation process andsubsequent analyses were performed in triplicate for each strain.

2.3. Viability of encapsulated lactic acid bacteria

The number of viable cells in the prepared microparticles was as-sessed shortly after the encapsulation process occurred, according toCoghetto, Brinques, and Ayub (2016), with modifications. To determinethe viability of the bacteria in the microparticles, 1 mL of a micro-particle suspension was mixed with 9mL phosphate buffer (100mM,pH 7.0) and shaken for 15min at 180 rpm in an orbital shaker untilcomplete release of the lactic acid bacteria. The resulting bacterialsuspension was serially diluted in 0.1% peptone water (w/v), and ali-quots of these dilutions were plated using the drop plate technique onMRS Agar. The Petri dishes were incubated at 37 °C for 48 h. In addi-tion, prior to the encapsulation process, the bacterial count in 1mL offeed solution was assessed using the same dilution, seeding and in-cubation conditions described above. L. plantarum ATCC8014, L. para-casei ML33 and L. pentosus ML82 survival is expressed as log CFU mL−1

(log of colony forming units per milliliter).

2.4. Viability of encapsulated lactic acid bacteria during refrigerated storage

M.WAP and M.PAP samples of L. plantarum ATCC8014, L. paracaseiML33 and L. pentosus ML82 were stored at 4 °C in phosphate buffer(10mM, pH 7.0) and periodically assessed for the survival of the en-capsulated bacteria over three months. The viability of the bacteria inthe microparticles was assessed according to Section 2.3. For thatpurpose, 1 mL of the microparticle suspension was mixed with 9mLphosphate buffer (100mM, pH 7.0) and shaken for 15min at 180 rpmin an orbital shaker to achieve the complete release of the lactic acidbacteria. The resulting bacterial suspension was serially diluted in 0.1%peptone water (w/v), and aliquots of these dilutions were plated usingthe drop plate technique on MRS Agar. The Petri dishes were incubatedat 37 °C for 48 h. Unencapsulated samples (also termed free cells) werekept under the same conditions and subjected to a viability assessmentduring the same period.

2.5. Viability of encapsulated lactic acid bacteria in a simulatedgastrointestinal tract

L. plantarum ATCC8014, L. paracasei ML33 and L. pentosus ML82uncapsulated and encapsulated with WAP and PAP were exposed tosimulated gastrointestinal solutions shortly after the encapsulationprocess to determine the ability of the bacteria to survive acidic con-ditions and the presence of bile salts. The test was performed accordingto the method proposed by Meira, Helfer, Velho, Lopes, and Brandelli(2012) with modifications. A 1.0mL aliquot of each encapsulatedbacterial sample was exposed to 1.0 mL of simulated gastric and in-testinal juices. Gastric juice was prepared with 3.0mgmL−1 pepsinfrom porcine gastric mucosa and 0.5% (w/v) NaCl, adjusted to pH 2.0,2.5 or 3.0. Intestinal juice was prepared to contain 1.0 mgmL−1 pan-creatin and 0.5% (w/v) NaCl, adjusted to pH 8.0, with or without5.0 g L−1 of a 1:1 mixture of sodium cholate and sodium deoxycholate.

Next, both solutions were sterilized by filtration through 0.22-μmmembranes (Sartorius Stedim Biotech, GmbH, Goettingen, Germany).Viable cell counts were performed at time zero and after 3 and 4 h forthe simulated gastric tract and intestinal tract conditions, respectively.The results were expressed as log CFU mL−1. Furthermore, un-encapsulated samples of L. plantarum ATCC8014, L. paracasei ML33 andL. pentosus ML82 were exposed to the simulated gastrointestinal juicesand used as controls.

2.6. Acidification potential of encapsulated lactic acid bacteria

Each M.WAP and M.PAP sample of L. plantarum ATCC8014, L.paracasei ML33 and L. pentosus ML82, as well as unencapsulated lacticacid bacteria, was added to 100mL of whole milk, which was kept at37 °C until reaching pH 4.6 (Krunić et al., 2015; Marafon, Sumi,Alcantara, Tamime, & De Oliveira, 2011). The acidification potential ofthe encapsulated and unencapsulated lactic acid bacteria was mon-itored by periodic pH measurements (Digimed DM-22, São Paulo,Brazil). The following kinetic parameters of acidification were assessed:Vmax (maximum rate of acidification, in units of pH per minute (up-H.min−1)); tVmax (time in hours to reach Vmax); pHmax (pH value cor-responding to the Vmax); and ΔpH (pH variation between the beginningand end of the incubation and pHmax).

2.7. Morphological characterization of the microparticles

The morphology of the microparticles was determined immediatelyafter they were made. The samples were stained and visualized underan optical microscope (Carl Zeiss model Primo Star, Jena, Germany) at100× magnification. Scanning electron microscopy (SEM) micro-photographs were also taken (Carl Zeiss model LS10, Jena, Germany)by first fixing the samples on double-sided carbon tape, which weredried at 30 °C for 3 h. Subsequently, the microparticles were coatedwith gold in a sputter coater (Q150R ES – Quorum Technologies, EastSussex, UK) and visualized by SEM in high vacuum mode to examinethe microparticle surface morphology and to estimate their size.

2.8. Data analysis

Analyses were performed in triplicate, and the results of en-capsulation assays were calculated as the average of three independentexperiments. The results for the viability of microorganisms, the en-capsulation conditions, the viability conditions of the unencapsulatedand encapsulated microorganisms during refrigerated storage and thesimulated GIT conditions, and the kinetic parameters of acidification ofthe unencapsulated and encapsulated microorganisms were subjectedto statistical analysis. The data were normally distributed and had equalvariance and were therefore evaluated using analysis of variance(ANOVA). The significance of the model was verified with the F test. Inthe significant models, the averages were compared with Tukey's test,with a significance level of 95% (p≤ .05) using BioEstat 5.3.

3. Results and discussion

3.1. Viability of encapsulated lactic acid bacteria

In this study, WAP and PAP combinations were assessed with re-spect to their ability to protect bacterial isolates with probiotic poten-tial during and after encapsulation by extrusion with vibrational tech-nology. The microorganism viability findings before and after theencapsulation process are outlined in Table 1.

The observed reduction in viable cells after the encapsulation pro-cess ranged from 0.07 to 0.74 log CFUmL−1, indicating that the use ofdifferent dairy by-products combined with alginate-pectin can protectthe microorganisms during the encapsulation process without affectingthe cell viability and maintaining a high count of isolates (≈

C. Eckert et al. Food Research International 113 (2018) 65–73

67

9 CFUmL−1).The extrusion method is arguably the least harmful to microbial

cells because it involves dripping a mixture of a hydrocolloid containingthe microorganism into an ionotropic solution. Vibration technology,which is an improvement of the extrusion technique, has been shown topreserve sensitive cells subjected to adverse conditions (Coghetto,Brinques, & Ayub, 2016; De Prisco et al., 2015; Shi et al., 2013;Whelehan & Marison, 2011). However, the protection of encapsulatedmicroorganisms by vibration technology requires the use of appropriatewall materials. Thus, the development of new microparticle productionapproaches is necessary to decrease their porosity and facilitate thecontrolled released of their content. Encapsulation using wall materialsthat form a dense protective network reduces the rate of diffusion of themedium into the microparticle. This effect reduces the exposure of themicroorganism sensitive to the adverse environments to which it will beexposed, increasing the protective effect of the encapsulation (Shi et al.,2013).

Sodium alginate is the most used biopolymer for this method, andthis material only partially protects the sensitive encapsulated material,particularly under acidic conditions requiring additional components toimprove the stability of the microcapsules (Shi et al., 2013). Therefore,several studies have searched for an adequate combination of alginateand other biopolymers to protect the material of interest (Belščak-Cvitanović et al., 2015; Chew, Tan, Long, & Nyam, 2015; Pankasemsuket al., 2016; Sandoval-Castilla et al., 2010).

When combined, alginate and pectin form a polyelectrolyte complexwith synergistic effect that produces gels at low pH values, which areobtained in media containing lactic acid bacteria due to the lactic acidproduced by these microorganisms. The complex formed allows chainlinkage between the hydrogels after the addition of divalent cations (forexample, Ca2+), improving the chemical and mechanical stability ofalginate and the encapsulation efficiency (Chew et al., 2015; Coghettoet al., 2016; Sandoval-Castilla et al., 2010).

Furthermore, particles produced by ionotropic gelation are usuallyporous, and the combination with other materials with emulsifyingeffects further strengthens the barrier imposed by the encapsulation.That is the case of whey, which is a promising wall material for themicroencapsulation of probiotic microorganisms. Cheese whey has ahigh nutritional value and is a source of high biological-value proteinsthat have an emulsifying effect that helps reduce the porosity and dif-fusion of the encapsulate. The permeate, obtained from the ultrafiltra-tion of the serum, is rich in lactose, which is incorporated into the al-ginate and pectin sugar chains, improving the stability of the wallmaterial formed (El-Salam & El-Shibiny, 2015; Young, Sarda, &Rosenberg, 1993). Thus, the proteins and lactose contained in the whey

have a gelling effect and, when combined with the hydroxides applied,increase the density of the network formed in the encapsulate, reducingthe diffusion rate of the microparticles and providing protection to thecells (Gebara et al., 2013; Prasanna & Charalampopoulos, 2018; Shiet al., 2013).

Few published studies have used this approach for encapsulation.However, they all describe its positive effect on the protection of sen-sitive materials, including De Prisco et al. (2015), who encapsulated L.reuteri DSM17938 in alginate and chitosan-alginate. The authors did notobserve differences in the viability of microorganisms after processing,obtaining a high encapsulation yield (> 92%). In addition, Shi et al.(2013) encapsulated L. bulgaricus in alginate-milk spheres. After thevibration extrusion process, the number of encapsulated cells washigher than 9 log CFUmL−1, with an observed decrease of 0.05–0.20log, similar to that of the present study.

Table 1Viability of bacterial strains (L. plantarum ATCC8014, L. paracasei ML33 and L.pentosus ML82) encapsulated with whey or permeate, alginate and pectin, be-fore and after being encapsulated.

Microparticles Number of viable cells

Before encapsulation(log CFU mL−1)

After encapsulation(log CFU mL−1)

Log reduction

M.WAP L.plantarum

9.66 (0.17) 9.22 (0.10) 0.44 (0.07)a,b

M.WAP ML33 9.47 (0.17) 9.36 (0.26) 0.11 (0.08)b

M.WAP ML82 9.96 (0.30) 9.22 (0.16) 0.74 (0.01)a,c

M.PAP L.plantarum

9.24 (0.29) 9.13 (0.18) 0.21 (0.06)b,c

M.PAP ML33 9.48 (0.01) 9.34 (0.20) 0.07 (0.02)b

M.PAP ML82 9.77 (0.40) 9.25 (0.26) 0.53 (0.01)a,c

Means ± standard deviation (n=3) with different superscript letters in thesame column indicating significant differences (p≤ .05). M.WAP:Microparticles Whey-Alginate-Pectin; M.PAP: Microparticles Permeate-Alginate-Pectin.

5

6

7

8

9

10

11

0 7 14 21 28 35 42 49 56 63 70 77 84 91

Via

ble

cell

(log

CFU

.mL-1

)

Days

(a)

5

6

7

8

9

10

11

0 7 14 21 28 35 42 49 56 63 70 77 84 91

Via

ble

cell

(log

CFU

.mL-1

)

Days

(b)

5

6

7

8

9

10

11

0 7 14 21 28 35 42 49 56 63 70 77 84 91

Via

ble

cell

(log

UFC

.mL-1

)

Days

(c)

Fig. 1. Viability of encapsulated strains (log CFUmL−1) during three months ofstorage at 4 °C. (a) L. plantarum ATCC8014; (b) L. paracaseiML33; (c) L. pentosusML82. M.WAP: Microparticles Whey-Alginate-Pectin; M.PAP:Microparticles Permeate-Alginate-Pectin; F.: Free cell.Means± standard deviation (n=3).

C. Eckert et al. Food Research International 113 (2018) 65–73

68

3.2. Viability of encapsulated lactic acid bacteria after refrigerated storage

All microparticles presented cell viabilities of> 6 log CFUmL−1

throughout the three-month storage period (Fig. 1). However, com-pared to the unencapsulated microorganism, the protective effect(p≤ .05) of both combinations of wall materials evaluated was pro-vided to L. plantarum ATCC8014. The encapsulates of the endogenousstrains, L. paracasei ML33 and L. pentosus ML82, for both combinationsof wall materials evaluated, did not present a significant protectiveeffect (p > .05) during the storage period, and thus new tests usingother wall materials are necessary for these microorganisms.

Nevertheless, the viability of microorganisms in microparticles re-presents a food supplementation alternative because they meet the re-quired concentration of probiotics (106–108 CFU g−1 product) thatprovides health benefits to consumers (FAO/WHO, 2002). Furthermore,the encapsulating materials are all safe for use in food and contribute topromoting health (Burgain et al., 2011; Martín et al., 2015).

An important factor in maintaining the viability of the encapsulatedmicroorganism during storage is its interaction with the encapsulatingmaterials used. The surface charge and hydrophobicity of bacteria areinvolved in the interaction between the proteins and the sugars thatmake up these materials, which is strain-specific and can be affected bythe components of the wall materials and the pH values of the mediumto which they are exposed (El-Salam & El-Shibiny, 2015). In addition,the whey used as wall materials is a natural habitat of these lacticbacteria, making the microparticles an environment with physico-chemical and biological characteristics suitable for the maintenance ofthese microorganisms.

In general, the WAP combination efficiently protected the micro-organisms, most likely due to the cheese whey composition providingproteins that reinforce the network formed between the polymers used,increasing the resistance of the microparticles (Shi et al., 2013). Com-paring the viability of encapsulated and unencapsulated microorgan-isms, L. plantarum ATCC8014 and L. pentosus ML82 were most pro-tected, with the microparticles showing twice the viability, on average,at the end of the storage period. The isolate L. paracasei ML33 showed adecrease of approximately 1 log cycle in encapsulated bacterial cellviability up to the 56th day of storage, matching the unencapsulatedbacterial cell viability on the 91st day.

Similar storage results have been reported in other studies, althoughthey were performed over a shorter study period. In a study by Shi et al.(2013), L. bulgaricus was encapsulated in alginate-milk spheres, and itssurvival was assessed for 30 days. The authors reported that over thisperiod, the microparticles showed no loss in cell viability, maintainingthe initial cell count of 10 log CFU g−1. Conversely, unencapsulatedcells showed a reduction of 7.7 log CFU g−1, indicating that the algi-nate-milk combination was essential in maintaining the viability of themicroorganism. Similarly, Sathyabama and Vijayabharathi (2014) en-capsulated Staphylococcus succinus (MAbB4) and Enterococcus faecium(FIdM3) with sodium alginate mixed with two prebiotic agents andassessed the survival of these encapsulated bacteria over 35 days at4 °C. The encapsulated bacteria had a smaller loss in viability (10%)compared to unencapsulated microorganisms (25%), indicating theneed for combining alginate with other encapsulating agents to im-prove the storage stability of the encapsulated microbial cells.

3.3. Viability of encapsulated lactic acid bacteria in the simulatedgastrointestinal tract

To have beneficial effects for the host, microorganisms must with-stand the adverse conditions of the GIT, which can be accomplished bythe encapsulation method. The results (Figs. 2 and 3) showed thatunencapsulated microorganisms were more sensitive to the simulatedgastric conditions (Fig. 2.a), being incubated with 3mgmL−1 pepsinand exposed to pH 2, as none of the isolates survived these conditions.Some strains of Lactobacillus are unable to survive low pH values

because these conditions inhibit the metabolic activity of the micro-organism, thereby reducing their viability (Sultana et al., 2000). Incontrast, encapsulation was effective in protecting bacterial cells, ap-proximately reducing all wall materials by 3 log cycles, maintainingviability above the requirements for use in probiotic foods (6 log CFUmL−1).

After being exposed to gastric tract conditions at a pH of 2.5 or 3(Fig. 2 b-c), a decrease of 3 and 2 log cycles was observed, respectively,when compared to the initial cell count, i.e., before exposure of theencapsulated microorganisms to the simulated GIT conditions.

A number of studies have reported that most probiotic micro-organisms do not withstand acidic stomach conditions. Thus, due to the

Fig. 2. Viability of encapsulated strains (log CFU mL−1) during sequential ex-posure to simulated gastric juice for 180min. (a) pH 2, (b) pH 2.5 and (c) pH 3.

Free cell, M.WAP: Microparticles Whey-Alginate-Pectin, M.PAP:Microparticles Permeate-Alginate-Pectin, a-d Means± standard deviation(n= 3) with different superscript letters in the same graph indicating sig-nificant differences (p < .05). ND=not detected.

C. Eckert et al. Food Research International 113 (2018) 65–73

69

presence of proteins with techno-functional properties, such as theability to form gels and emulsions, whey are an excellent encapsulatingmatrix when combined with alginate and pectin. The gelling effect ofthis combination is an excellent barrier to low pH, increasing the sur-vival of microorganisms encapsulated in hydrocolloid matrices withwhey. That occurs because the dense hydrogel network formed reducesthe diffusion rate into the microparticle, thereby reducing the exposureof the microorganisms to the acid medium to which they are exposed(Doherty et al., 2011; Guérin, Vuillemard, & Subirade, 2003; Prasanna& Charalampopoulos, 2018; Shi et al., 2013).

To simulate the intestinal tract (Fig. 3), samples were incubatedwith 1mgmL−1 of pancreatin at pH 8, with or without the addition of

0.5% (w/v) bile salts. When compared to the initial cell count, in theabsence of bile salts (Fig. 3.a), a reduction of 1 log cycle was observed,with no significant differences between unencapsulated and en-capsulated microbial cells. In contrast, in the presence of 0.5% (w/v)bile salts (Fig. 3.b), a more pronounced reduction of 4 log cycles wasobserved.

Overall, the WAP combination had a stronger protective effectagainst the simulated GIT than PAP. These results are most likely due tothe physicochemical composition of the by-products used as en-capsulating materials and the interaction between them in the formedmicroparticles. It should be noted that under both gastric and intestinalconditions, the survival rates of microorganisms indigenous to thesouthern region of Brazil (L. paracaseiML33 and L. pentosusML82) weresimilar to those of L. plantarum ATCC8014, indicating that these strainshave potential for being encapsulated and used in foods. The protectionprovided by the encapsulation under the simulated GIT conditions maybe due to the interaction of the metabolic activity of microorganismsand wall materials used, or due to the natural resistance of these bac-teria to different pH and digestive enzymes (Gebara et al., 2013).

Zaeim et al. (2017) encapsulated L. plantarum ATCC8014 with al-ginate in chitosan-CaCl2 and in CaCl2 followed by coating with chitosanby electrospray. When exposed to the simulated gastrointestinal tract,the microcapsules made using chitosan-CaCl2 showed a reduction of 3.4log, whereas no microorganisms survived in the microcapsules formedusing CaCl2. Similarly, Ding and Shah (2007) encapsulated eight strainsof probiotic bacteria (Lactobacillus and Bifidobacterium) with alginate byemulsion. The microparticles exposed to the simulated GIT showed areduction in viability of 4 log cycles in the gastric tract and 3 log cyclesin the intestinal tract. The results of these studies were lower than thefindings of the present study, which may be related to the choice ofencapsulating materials, to the encapsulation technique used or to theresistance of the bacterial strains to adverse conditions.

3.4. Acidification potential of encapsulated lactic acid bacteria

Table 2 outlines the results from the acidification potential of milkat 37 °C by the unencapsulated and encapsulated lactic acid bacteria.The observed Vmax using unencapsulated microorganisms ranged from27.09 to 31.09 (×10−4) upH.min−1, which was significantly higher(p≤ .05) than that of encapsulated microorganisms (Vmax ranging from10.05 to 13.45 (×10−4) upH.min−1). This parameter is related to thefermentation time (tVmax) required for the milk pH to reach 4.6, whichwas lower in the unencapsulated samples (6 h) than in the encapsulatedsamples (23–27 h). These results indicate that all wall materials assayedprovided a barrier that hindered substrate diffusion in the micro-particles, affecting the activity of the L. plantarum ATCC8014, L. para-casei ML33 and L. pentosus ML82 isolates and affecting the milk fer-mentation period.

a a

a a a a a

a a

0

1

2

3

4

5

6

7

8

9

L. plantarum ML33 ML82

Via

ble

cell

(log

CFU

.mL

-1)

Strain

(a)

a,b,c,d,e b,d c,d,e

a,c a

d b,c,d a,d a,e

0

1

2

3

4

5

6

7

8

9

10

L. plantarum ML33 ML82

Via

ble

cell

(log

CFU

.mL

-1)

Strain

(b)

Fig. 3. Viability of encapsulated strains (log CFU mL−1) during sequential ex-posure to simulated intestinal juice for 240min. (a) Without bile salt and (b)with 0.5% (w/v) bile salt. Free cell, M.WAP: Microparticles Whey-Alginate-Pectin, M.PAP: Microparticles Permeate-Alginate-Pectin. a-e

Means± standard deviation (n= 3) with different superscript letters in thesame graph indicating significant differences (p < .05).

Table 2Kinetic acidification parameters of free and encapsulated strains in milk fermented at 37 °C until reaching a pH of 4.6.

Samples Kinetic acidification parameters

Vmax (×10−4 upH.min−1) tVmax (h) pHmax ΔpH

M.WAP L. plantarum 12.20 (0.30)b 23 (0.00) 4.68 (0.28) 1.80 (0.20)M.WAP ML33 12.60 (0.33)b 23 (0.00) 4.64 (0.24) 1.88 (0.32)M.WAP ML82 11.65 (0.35)b 23 (0.00) 4.65 (0.05) 1.75 (0.05)

M.PAP L. plantarum 10.05 (0.05)b 27 (0.00) 4.63 (0.01) 1.62 (0.01)M.PAP ML33 13.45 (0.15)b 23 (0.00) 4.35 (0.15) 1.85 (0.01)M.PAP ML82 11.00 (0.10)b 26 (0.00) 4.66 (0.05) 1.71 (0.01)F.L. plantarum 28.05 (0.27)a 6 (0.00) 4.57 (0.09) 1.81 (0.10)F.ML33 31.90 (0.30)a 6 (0.00) 4.58 (0.01) 1.85 (0.09)F.ML82 27.90 (0.10)a 6 (0.00) 4.51 (0.04) 1.80 (0.03)

Means ± standard deviation (n= 3) with different superscript letters in the same column indicating significant differences (p≤ .05). M.WAP: Microparticles Whey-Alginate-Pectin; M.PAP: Microparticles Permeate-Alginate-Pectin; F. Free cell.

C. Eckert et al. Food Research International 113 (2018) 65–73

70

Krunić et al. (2015) immobilized a starter culture (80% Strepto-coccus salivarius subsp. thermophilus, 13% L. acidophilus, 6% B. bifidumand 1% L. delbrueckii subsp. bulgaricus) in alginate and chitosan mi-croparticles, which were added to milk and assessed with respect to theacidification profile. The fermentation time of the unencapsulatedculture starter was 1.5 h shorter than that of the encapsulated culture.In the present study, the difference in fermentation time between un-encapsulated and encapsulated cells was greater, approximately 18 h.In contrast, Marafon et al. (2011) studied the kinetic parameters ofacidification of probiotic yogurts containing S. thermophilus, L. del-brueckii subs. Bulgaricus and B. lactis subs. Animalis supplemented withprotein from dairy by-products (skimmed milk powder, whey proteinconcentrate and sodium caseinate). For all supplemented milk bases,

the acidification time was 6 h, a result that was similar to that of un-encapsulated isolates added to milk in this study. Thus, the decreasedacidification potential of encapsulated lactic acid bacteria may be at-tributed to the encapsulation, which reduces the rate of fermentationand consequently increases the time required for milk products to reachthe pH corresponding to the isoelectric point of casein.

3.5. Morphological characterization of microparticles

Optical microscopy and SEM images of the microparticles of bac-teria encapsulated with WAP and PAP are shown in Figs. 4 and 5, re-spectively. All wall materials tested produced uniform and reproduciblemicroparticles with different morphologies depending on the whey

Fig. 4. Optical microscopy images of encapsulated strains obtained using vibration technology: (a) M.WAP: Microparticles Whey-Alginate-Pectin, 100× and (b)M.PAP: Microparticles Permeate-Alginate-Pectin, 100×.

C. Eckert et al. Food Research International 113 (2018) 65–73

71

used. This is the result of the encapsulation technology used, in whichthe vibration applied on the laminar jet is responsible for promoting theuniform and reproducible break-up of the jet, due to the application of apermanent force of defined frequency, resulting in the formation of oneparticle per hertz of frequency (Whelehan & Marison, 2011).

Cheese whey generated spherical microparticles (Figs. 4.a and 5.a),while whey permeate microparticles were drop shaped (Figs. 4.b and5.b). This difference is likely a result of the viscosity of the feed solutionused, which depends on the combination of wall materials used forencapsulation (Nemethova, Lacik, & Razga, 2015; Whelehan & Marison,2011).

The expected size of the microparticles formed using both combi-nations of wall materials was in the order of μm (~160 μm), twice thediameter of the feed nozzle used in this experiment (BÜCHI–Encapsulator B-395 Pro Datasheet), as determined by the optical mi-croscopy images of the microparticles (Fig. 4). However, for improvedvisualization, SEM images of dried microparticles (Fig. 5) were ac-quired, in which the microcapsule diameter decreased to approximately100 μm due to water loss. Considering the size of the prepared micro-particles, they are suitable for being added into foods, because smallerparticles ensure a homogeneous and quality product without affectingthe sensory properties (Arslan et al., 2015; Martín et al., 2015). Moststudies using extrusion by vibrational technology produce micro-capsules with larger diameters, which may be a negative factor for theirfood applicability (Del Gaudio et al., 2005; Martoni et al., 2008;Mazzitelli et al., 2008; Nemethova et al., 2015; Shi et al., 2013).

4. Conclusion

In this study, extrusion by vibrational technology was a suitablemethod for the encapsulation of lactic acid bacteria. The WAP wallmaterial improved the stability of the microcapsule and protected thesensitive cells. During the refrigerated storage period, the encapsulationwas effective in protecting the microorganism L. plantarum ATCC8014,however it was not effective for the strains endogenous to the southernregion of Brazil (L. paracasei ML33 and L. pentosus ML82), thus re-quiring that other wall materials that may provide a better protectiveeffect be tested. In the simulated GIT conditions, wall materials con-taining cheese whey showed improved protection of encapsulated mi-croorganisms. The microparticles had a lower milk acidification ratethan unencapsulated microorganisms, affecting the fermentation profileof the product. The indigenous lactic acid bacteria showed a similarbehavior to L. plantarum ATCC8014, indicating the potential of theseautochthonous isolates. Lastly, the prepared microparticles can be usedin foods due to the adequate concentration of viable microbial cellspresent and their morphological characteristics.

Acknowledgements

We would like to thank Launer Química Ltd. for donating the lacticacid bacterial strains L. paracaseiML33 and L. pentosusML82. We wouldlike to acknowledge Conselho Nacional de Desenvolvimento Científicoand Tecnológico (CNPq) and Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES) for scholarships. We would also liketo thank Tecnovates, Universidade do Vale do Taquari - Univates, andSecretaria do Desenvolvimento Econômico, Ciência and Tecnologia doRio Grande do Sul (SDECT) for their financial support.

References

Anal, A. K., & Singh, H. (2007). Recent advances in microencapsulation of probiotics forindustrial applications and targeted delivery. Trends in Food Science & Technology,18(5), 240–251.

Arslan, S., Erbas, M., Tontul, I., & Topuz, A. (2015). Microencapsulation of probioticSaccharomyces cerevisiae var. boulardii with different wall materials by spray drying.LWT-Food Science and Technology, 63(1), 685–690.

Bekhit, M., Sánchez-González, L., Messaoud, G. B., & Desobry, S. (2016). Encapsulation ofLactococcus lactis subsp. lactis on alginate/pectin composite microbeads: Effect ofmatrix composition on bacterial survival and nisin release. Journal of FoodEngineering, 180, 1–9.

Belščak-Cvitanović, A., Komes, D., Karlović, S., Djaković, S., Špoljarić, I., Mršić, G., &Ježek, D. (2015). Improving the controlled delivery formulations of caffeine in al-ginate hydrogel beads combined with pectin, carrageenan, chitosan and psyllium.Food Chemistry, 167, 378–386.

Burgain, J., Gaiani, C., Linder, M., & Scher, J. (2011). Encapsulation of probiotic livingcells: From laboratory scale to industrial applications. Journal of Food Engineering,104(4), 467–483.

Carvalho, F., Prazeres, A. R., & Rivas, J. (2013). Cheese whey wastewater:Characterization and treatment. Science of the Total Environment, 445, 385–396.

Chew, S. C., Tan, C. P., Long, K., & Nyam, K. L. (2015). In-vitro evaluation of kenaf seedoil in chitosan coated-high methoxyl pectin-alginate microcapsules. Industrial Cropsand Products, 76, 230–236.

Coghetto, C. C., Brinques, G. B., & Ayub, M. A. Z. (2016). Probiotics production andalternative encapsulation methodologies to improve their viabilities under adverseenvironmental conditions. International Journal of Food Sciences and Nutrition, 67(8),929–943.

Coghetto, C. C., Brinques, G. B., Siqueira, N. M., Pletsch, J., Soares, R. M. D., & Ayub, M.A. Z. (2016). Electrospraying microencapsulation of Lactobacillus plantarum enhancescell viability under refrigeration storage and simulated gastric and intestinal fluids.Journal of Functional Foods, 24, 316–326.

De Prisco, A., Maresca, D., Ongeng, D., & Mauriello, G. (2015). Microencapsulation byvibrating technology of the probiotic strain Lactobacillus reuteri DSM 17938 to en-hance its survival in foods and in gastrointestinal environment. LWT-Food Science andTechnology, 61(2), 452–462.

De Vos, P., Faas, M. M., Spasojevic, M., & Sikkema, J. (2010). Encapsulation for pre-servation of functionality and targeted delivery of bioactive food components.International Dairy Journal, 20(4), 292–302.

Del Gaudio, P., Colombo, G., Colombo, P., Russo, P., & Sonvico, F. (2005). Mechanisms offormation and disintegration of alginate beads obtained by prilling. InternationalJournal of Pharmaceutics, 302(1), 1–9.

Desai, K. G. H., & Jin Park, H. (2005). Recent developments in microencapsulation of foodingredients. Drying Technology, 23(7), 1361–1394.

Ding, W. K., & Shah, N. P. (2007). Acid, bile, and heat tolerance of free and micro-encapsulated probiotic bacteria. Journal of Food Science, 72(9), M446–M450.

Doherty, S. B., Gee, V. L., Ross, R. P., Stanton, C., Fitzgerald, G. F., & Brodkorb, A. (2011).Development and characterisation of whey protein micro-beads as potential matricesfor probiotic protection. Food Hydrocolloids, 25(6), 1604–1617.

El-Salam, M. H. A., & El-Shibiny, S. (2015). Preparation and properties of milk proteins-

Fig. 5. SEM images of encapsulated strains obtained using vibration technology: (a) M.WAP: Microparticles Whey-Alginate-Pectin, 15,000× and (b) M.PAP:Microparticles Permeate-Alginate-Pectin, 15,000×.

C. Eckert et al. Food Research International 113 (2018) 65–73

72

based encapsulated probiotics: A review. Dairy Science & Technology, 95(4), 393–412.FAO/WHO (2002). Guidelines for the evaluation of probiotics in food. Report of a Joint FAO/

WHO working group on drafting guidelines for the evaluation of probiotics in food.London, Ontario, Canada: Food and Agriculture Organization of the United Nations/World Health Organization.

Gebara, C., Chaves, K. S., Ribeiro, M. C. E., Souza, F. N., Grosso, C. R., & Gigante, M. L.(2013). Viability of lactobacillus acidophilus La5 in pectin–whey protein micro-particles during exposure to simulated gastrointestinal conditions. Food ResearchInternational, 51(2), 872–878.

Guérin, D., Vuillemard, J. C., & Subirade, M. (2003). Protection of bifidobacteria en-capsulated in polysaccharide-protein gel beads against gastric juice and bile. Journalof Food Protection, 66(11), 2076–2084.

Hugo, A. A., Bruno, F., & Golowczyc, M. A. (2016). Whey permeate containing galacto-oligosaccharides as a medium for biomass production and spray drying ofLactobacillus plantarum CIDCA 83114. LWT-Food Science and Technology, 69, 185–190.

JAY, J. M. (2000). Modern food microbiology. 6 ed. Maryland. Aspen Publishers.Kent, R. M., & Doherty, S. B. (2014). Probiotic bacteria in infant formula and follow-up

formula: Microencapsulation using milk and pea proteins to improve microbiologicalquality. Food Research International, 64, 567–576.

Keogh, M. K., & O'Kennedy, B. T. (1999). Milk fat microencapsulation using whey pro-teins. International Dairy Journal, 9(9), 657–663.

Khem, S., Small, D. M., & May, B. K. (2016). The behaviour of whey protein isolate inprotecting Lactobacillus plantarum. Food Chemistry, 190, 717–723.

Krunić, T.Ž., Bulatović, M. L., Obradović, N. S., Vukašinović-Sekulić, M. S., & Rakin, M. B.(2015). Effect of immobilisation materials on viability and fermentation activity ofdairy starter culture in whey-based substrate. Journal of the Science of Food andAgriculture, 96(5), 1723–1729.

Livney, Y. D. (2010). Milk proteins as vehicles for bioactives. Current Opinion in Colloid &Interface Science, 15(1), 73–83.

Marafon, A. P., Sumi, A., Alcantara, M. R., Tamime, A. Y., & De Oliveira, M. N. (2011).Optimization of the rheological properties of probiotic yoghurts supplemented withmilk proteins. LWT-Food Science and Technology, 44(2), 511–519.

Maresca, D., De Prisco, A., La Storia, A., Cirillo, T., Esposito, F., & Mauriello, G. (2016).Microencapsulation of nisin in alginate beads by vibrating technology: Preliminaryinvestigation. LWT-Food Science and Technology, Vol. 66, 436–443.

Martín, M. J., Lara-Villoslada, F., Ruiz, M. A., & Morales, M. E. (2015).Microencapsulation of bacteria: A review of different technologies and their impacton the probiotic effects. Innovative Food Science & Emerging Technologies. Vol. 27.Innovative Food Science Emerging Technologies (pp. 15–25).

Martoni, C., Bhathena, J., Urbanska, A. M., & Prakash, S. (2008). Microencapsulated bilesalt hydrolase producing Lactobacillus reuteri for oral targeted delivery in the gas-trointestinal tract. Applied Microbiology and Biotechnology, 81(2), 225–233.

Mazzitelli, S., Tosi, A., Balestra, C., Nastruzzi, C., Luca, G., Mancuso, F., ... Calvitti, M.(2008). Production and characterization of alginate microcapsules produced by avibrational encapsulation device. Journal of Biomaterials Applications, 23(2), 123–145.

Meira, S. M. M., Helfer, V. E., Velho, R. V., Lopes, F. C., & Brandelli, A. (2012). Probioticpotential of Lactobacillus spp. isolated from Brazilian regional ovine cheese. Journal ofDairy Research, 79(01), 119–127.

Mollea, C., Marmo, L., & Bosco, F. (2013). Valorisation of cheese whey, a by-product fromthe dairy industry. Food industry. InTech Open Access Publisher.

Naqash, F., Masoodi, F. A., Rather, S. A., Wani, S. M., & Gani, A. (2017). Emergingconcepts in the nutraceutical and functional properties of pectin− a review.Carbohydrate Polymers, 168, 227–239.

Nemethova, V., Lacik, I., & Razga, F. (2015). Vibration technology for microencapsula-tion: The restrictive role of viscosity. Journal of Bioprocessing & Biotechniques,5(1), 1–3.

Pankasemsuk, T., Apichartsrangkoon, A., Worametrachanon, S., & Techarang, J. (2016).Encapsulation of Lactobacillus casei 01 by alginate along with hi-maize starch forexposure to a simulated gut model. Food Bioscience, 16, 32–36.

Prasanna, P. H. P., & Charalampopoulos, D. (2018). Encapsulation of Bifidobacteriumlongum in alginate-dairy matrices and survival in simulated gastrointestinal condi-tions, refrigeration, cow milk and goat milk. Food Bioscience, 21, 72–79.

Ribeiro, M. C. E., Chaves, K. S., Gebara, C., Infante, F. N., Grosso, C. R., & Gigante, M. L.(2014). Effect of microencapsulation of Lactobacillus acidophilus LA-5 on physico-chemical, sensory and microbiological characteristics of stirred probiotic yoghurt.Food Research International, 66, 424–431.

Sandoval-Castilla, O., Lobato-Calleros, C., García-Galindo, H. S., Alvarez-Ramírez, J., &Vernon-Carter, E. J. (2010). Textural properties of alginate–pectin beads and survi-vability of entrapped Lb. casei in simulated gastrointestinal conditions and in yoghurt.Food Research International, 43(1), 111–117.

Santos, T. T., Ornellas, R. M. S., Arcucio, L. B., Oliveira, M. M., Nicoli, J. R., Dias, C. V., &Vinderola, G. (2016). Characterization of lactobacilli strains derived from cocoafermentation in the south of Bahia for the development of probiotic cultures. LWT-Food Science and Technology, 73, 259–266.

Sathyabama, S., & Vijayabharathi, R. (2014). Co-encapsulation of probiotics with pre-biotics on alginate matrix and its effect on viability in simulated gastric environment.LWT-Food Science and Technology, 57(1), 419–425.

Shi, L. E., Li, Z. H., Zhang, Z. L., Zhang, T. T., Yu, W. M., Zhou, M. L., & Tang, Z. X. (2013).Encapsulation of Lactobacillus bulgaricus in carrageenan-locust bean gum coated milkmicrospheres with double layer structure. LWT-Food Science and Technology, 54(1),147–151.

Smithers, G. W. (2008). Whey and whey proteins—From ‘gutter-to-gold. InternationalDairy Journal, 18(7), 695–704.

Smithers, G. W. (2015). Whey-ing up the options–yesterday, today and tomorrow.International Dairy Journal, 48, 2–14.

Soccol, C. R., Vandenberghe, L. P. D. S., Spier, M. R., Medeiros, A. B. P., Yamaguishi, C. T.,Lindner, J. D. D., ... Thomaz-Soccol, V. (2010). The potential of probiotics: A review.Food Technology and Biotechnology, 48(4), 413–434.

Su, L. C., Lin, C. W., & Chen, M. J. (2007). Development of an oriental-style dairy productcoagulated by microcapsules containing probiotics and filtrates from fermented rice.International Journal of Dairy Technology, 60(1), 49–54.

Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., & Kailasapathy, K.(2000). Encapsulation of probiotic bacteria with alginate–starch and evaluation ofsurvival in simulated gastrointestinal conditions and in yoghurt. International Journalof Food Microbiology, 62(1), 47–55.

Sybesma, W., Kort, R., & Lee, Y. K. (2015). Locally sourced probiotics, the next oppor-tunity for developing countries? Trends in Biotechnology, 33(4), 197–200.

Vanderpool, C., Yan, F., & Polk, D. B. (2008). Mechanisms of probiotic action:Implications for therapeutic applications in inflammatory bowel diseases.Inflammatory Bowel Diseases, 14(11), 1585–1596.

Vivek, K. (2013). Use of encapsulated probiotics in dairy based foods. International Journalof Food, Agriculture and Veterinary Sciences, 3(1), 188–199.

Voo, W. P., Ravindra, P., Tey, B. T., & Chan, E. S. (2011). Comparison of alginate andpectin based beads for production of poultry probiotic cells. Journal of Bioscience andBioengineering, 111(3), 294–299.

Whelehan, M., & Marison, I. W. (2011). Microencapsulation using vibrating technology.Journal of Microencapsulation, 28(8), 669–688.

Young, S. L., Sarda, X., & Rosenberg, M. (1993). Microencapsulating properties of wheyproteins. 2. Combination of whey proteins with carbohydrates. Journal of DairyScience, 76(10), 2878–2885.

Zaeim, D., Sarabi-Jamab, M., Ghorani, B., Kadkhodaee, R., & Tromp, R. H. (2017).Electrospray assisted fabrication of hydrogel microcapsules by single-and double-stage procedures for encapsulation of probiotics. Food and Bioproducts Processing, 102,250–259.

Zhang, Y., Lin, J., & Zhong, Q. (2016). S/O/W emulsions prepared with sugar beet pectinto enhance the viability of probiotic Lactobacillus salivarius NRRL B-30514. FoodHydrocolloids, 52, 804–810.

C. Eckert et al. Food Research International 113 (2018) 65–73

73