Journal of Food Engineeringstatic.tongtianta.site/paper_pdf/d2150bb6-bcf9-11e9-bcd6-00163e08… · Lactobacillus casei (2651 1951 RPK) was purchased from NCIM, Pune. Two separate

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Journal of Food Engineering

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

Controlled release of microencapsulated probiotics in food matrix

Soham Basua, Debolina Banerjeea, Ranjana Chowdhurya,∗, Pinaki Bhattacharyab

a Chemical Engineering Department, Jadavpur University, Kolkata 700032, Indiab Chemical Engineering Department, Heritage Institute of Technology, Kolkata 700107, India

A R T I C L E I N F O

Keywords:MicroencapsulationLactobacillus caseiConcentration profilesExponential growthBurst releaseFood matrix

A B S T R A C T

The present study focusses on direct entrapment, internal and external microencapsulation of Lactobacillus casei(L. casei) in bio-polymer (alginate) matrix and their performance in lactose-rich media. Probiotic beads andmicrocapsules were suspended in modified de Mann-Rogosa-Sharpe (MRS) medium containing lactose and arefreshing drink-green coconut water, fortified with the same carbohydrate. A novel mathematical model hasbeen developed to simulate substrate and biomass time-concentration profiles as a function of radial positionwithin the probiotic beads/capsules. Further, using the burst-release mechanism, the model could also predictthe release time of L. casei-868min, 587min and 574min from alginate beads, external and internal micro-capsules respectively. The predicted values were in good agreement with the experimental results for bothmodified MRS medium and coconut water. The release time of probiotics∼10 h indicates the scope for potentialapplication of probiotic beads/capsules in the production of probiotic functional health drinks using green co-conut water.

1. Introduction

A probiotic is a live microbial feed supplement, which beneficiallyaffects the host by improving its intestinal microbial balance (AFRC,1989; Nithya and Halami, 2013). The most commonly used probioticsinclude lactobacillus and bifidobacteria (Collado et al., 2008). Strepto-coccus species, Saccharomyces cerevisiae (Burgain et al., 2011), otherbacterium (Propionibacterium freudenreichii, Enterococcus, Escherichiacoli) (Gupta and Garg, 2009) are some of the various types of probioticsreported in literature. Probiotics, as reported in various studies, havebeen shown to be effective in the treatment of intestinal disorders(Kurmann, 1991), inflammatory diseases (Malchow, 1997), allergies(Majamaa and Isolauri, 1997) etc. They have been applied in a multi-tude of food matrices, enhancing their nutritional value and aiding intheir preservation (Mohammadi et al., 2011; Tripathi and Giri, 2014).To confer the above–mentioned beneficial effects, it is necessary thatthe probiotics survive a number of damaging influences during pro-cessing of their resident food matrices (Dias et al., 2017).

Microencapsulation is a technique by which the desired product isprotected from detrimental external influences and is achieved viaphysical/chemical/physicochemical routes (Champagne et al., 1994). Acommon biomaterial used for microencapsulation or immobilization ofprobiotic bacteria is alginate which has been used extensively in theconcentration range of 0.5–5%, as reported in various literature(Hansen et al., 2002; Kebary et al., 1998; Shah and Ravula, 2000).

External gelation and internal gelation are two of the most widely usedmethods of chemical emulsification/ionic gelation among which in-ternal gelation is the more preferred method due to its property ofproducing microspheres having very narrow size distribution (Chunet al., 2014; Cook et al., 2012). Microencapsulation has been applied ina wide variety of food matrices so as to reduce probiotic cell mortality(Chen et al., 2017) and increase their shelf–life (Anal and Singh, 2007;Banerjee et al., 2017a).

The present research group has investigated L. casei survival ingastro–intestinal tract, and burst-release of the encapsulated cells insynbiotic environment (Banerjee et al., 2017b). The present study fo-cusses on probiotic L. casei survival in food matrix; where externallysupplied substrate; i.e., lactose, diffuses into the alginate matrix. Pro-biotic growth is thereby a function of reaction–diffusion of substrate.This work investigates the mechanism and kinetics of probiotic growthin alginate matrix and the potential of microencapsulation as a tech-nique to increase the shelflife of functional foods. Although the utilityof microencapsulation in increasing the shelf-life of functional foods hasbeen stated in earlier journals (Anal and Singh, 2007; Banerjee et al.,2017a; Dias et al., 2017), the study of this mechanism is very importantand has not been attempted previously in any known literature.Moreover, the application of microencapsulated probiotic microorgan-isms in liquid food is mostly restricted to fruit and vegetable juices(Anekella and Orsat, 2013; Reid et al., 2007). To the best of ourknowledge, probiotic microcapsules have never been applied in

https://doi.org/10.1016/j.jfoodeng.2018.06.005Received 1 February 2018; Received in revised form 2 June 2018; Accepted 4 June 2018

∗ Corresponding author. Chemical Engineering Department, Jadavpur University, Jadavpur, Kolkata 700032, India.E-mail address: [email protected] (R. Chowdhury).

Journal of Food Engineering 238 (2018) 61–69

Available online 06 June 20180260-8774/ © 2018 Elsevier Ltd. All rights reserved.

T

mineral-rich natural health drinks, e.g., coconut water. However, co-conut water is a refreshing beverage, common in tropical and sub–-tropical regions, including south-east Asia, Pacific Islands, Africa andthe Caribbean (DebMandal and Mandal, 2011; Ganguly, 2013). Notonly does coconut water have natural hydrating qualities, it is also anatural source of electrolytes and is known to prevent myocardial dis-eases (Camargo Prado et al., 2015; Yong et al., 2009). It has even beenused for intravenous application (Campbell-Falck et al., 2000). It isunderstandable that green coconut–water rich in encapsulated L. caseishall have three–fold benefits: coconut water and probiotics shall beable to confer their individual beneficial effects upon the host and theenzymes released by the probiotics shall help in preservation of thecoconut water, which in its native form, gets spoiled easily. Thoroughinvestigation should also be made on the release of probiotics in such aunique nutrient–rich beverage. The release time for lactose basedmodified MRS medium will be compared with that obtained for lactose-rich coconut water. One novelty of the present research lies in thisaspect.

Under the present research study, a model based on re-action–diffusion will be developed to predict the availability of lactosewithin microcapsules formed through internal/external gelation andthrough entrapment in Ca-alginate beads; and to predict the con-centration profiles of cells suspended in lactose rich liquid medium(modified MRS) over their incubation periods. The “release time” ofprobiotic microorganisms will also be determined experimentally formicrocapsules and beads suspended in lactose rich modified MRSmedium. These experimental values of release time will be comparedwith the simulated values for validation of the model. The experimentshall finally be carried out using lactose–rich raw, green coconut wateras the suspension medium.

2. Materials and methods

2.1. Chemicals

Calcium chloride, sodium alginate (Na-alginate), Tween-80, aceticacid, glycerol, sodium dodecyl sulphate (SDS), beef extract, calciumcarbonate, lactose purchased from Merck, India were used. Yeast ex-tract, peptone, sodium acetate, di-potassium hydrogen phosphate, tri-ammonium citrate, magnesium sulphate, manganese sulphate, sodiumchloride, di-sodium hydrogen phosphate, potassium di-hydrogenphosphate, glucose purchased from Himedia, India, were used for thisstudy. Rice bran oil and green coconut water purchased from localmarket were used.

2.2. Microorganism

Lactobacillus casei (2651 1951 RPK) was purchased from NCIM,Pune. Two separate cultures of L. casei were prepared, one usingmodified MRS and the other using raw, green coconut water as growthmedium (Eratte et al., 2016).

2.3. Growth medium and other reagents

2.3.1. Composition of modified de Mann-Rogosa-Sharpe (MRS) mediumBeef extract: 10 g/L; yeast extract: 5 g/L; peptone: 10 g/L; sodium

acetate: 5 g/L; di-potassium hydrogen phosphate: 2 g/L; tri-ammoniumcitrate: 2 g/L; magnesium sulphate: 0.05 g/L; manganese sulphate:0.05 g/L and lactose: 20 g/L; pH-7.

2.3.2. Composition of green coconut waterThe composition of green coconut water was assumed to be the

same as that reported by Campos et al., 1996-soluble solids: 4.46–7.02Brix (at 20 °C); total titratable acidity: 13.9–76.8 mg citric acid/100mLand total reducing sugars: 4.90 ± 0.20 g/100mL. The normal pH ofgreen coconut water is 4.7–6.4.

2.3.3. Preparation of lactose rich green coconut waterLactose was added to fresh green coconut water to maintain a

concentration of 20 g/L and the pH was adjusted to 7 by the addition ofphosphate buffer solution of pH 8.

2.4. Preparation of alginate/L. casei beads/microcapsules

2.4.1. Beads prepared by direct entrapmentFor bead preparation by direct calcium alginate entrapment via

standard protocol, 10mL culture cells were mixed with 40mL 1%sterile Na–alginate solution. 10mL of the mixture was then added dropwise into a 1% CaCl2 solution at room temperature and stirred con-tinuously. The drop-wise addition was performed manually using amicropipette with the reading set at 0.5 mL (Same applies for sections2.4.2. and 2.4.3.). This was done so as to ensure uniformity in dischargevolume and thereby drop-size. Beads were formed via hardening fol-lowing which they were washed with saline water. The formed beadswere stored at 4 °C (Sheu and Marshall, 1993).

2.4.2. Beads prepared by external gelationFor synthesis of beads via external gelation, 1 mL of L. casei culture

was mixed with 4mL sodium alginate. 1 mL of the formed mixture wasstirred with 5mL vegetable oil. The resulting solution was mixed with0.2% Tween-80 and 0.25% SDS. This mixture was stirred at 200 rpm for30min. CaCl2 was added quickly but gently down the side of a beakerin which the mixture was taken. The formed beads were kept un-disturbed for 30min. They were then filtered with muslin cloth. Finally,the beads were washed with 0.9% saline water containing 0.05% gly-cerol. The washed beads were stored at 4 °C (Chan et al., 2006; Songet al., 2013).

2.4.3. Beads prepared by internal gelation4mL Na-alginate (2%) solution was mixed with 1mL of L. casei

culture and 0.08 g CaCO3 powder. 1 mL was taken from the mixture and5mL vegetable oil and 1mL Tween 80 were added to it. The resultingsolution was stirred at 300 rpm for 30min. Acetic acid was addeddropwise to the solution at 15–25 °C. Resulting beads were filtered withmuslin cloth and stored at 4 °C (Banerjee et al., 2017b; Holkem et al.,2017).

2.5. Determination of “burst-release” time for beads and microspheres

When the microcapsules are suspended in suitable medium, en-trapped cells grow in number. At a critical concentration of entrappedcells, the biotic phase occupies the entire core volume and consequentlythe beads or microspheres burst and all probiotic cells are released. Thistime may be defined as the “burst-release” time for beads and micro-spheres. For obtaining the “burst-release” time of the capsules synthe-sized via the aforementioned methods, they are at first suspended indifferent media, namely, lactose-rich modified MRS medium and greencoconut water (lactose concentration-20 g/L, net volume of media andbeads/microspheres-50 mL). The suspensions in the conical flask wereallowed to stand and were observed periodically so as to obtain thetimes at which the capsules burst. The bursting of the capsules wasindicated by the suspension medium inside the conical flask becomingturbid abruptly. This change in turbidity was profound and easilyperceptible to the naked eye.

2.6. Determination of growth kinetics of L. casei on lactose

Cell growth kinetic parameters of L. casei on lactose were obtainedusing the classical Monod type substrate uninhibited unstructuredmodel as has been described in Banerjee et al. (2017b). The pertinentequation is:

S. Basu et al. Journal of Food Engineering 238 (2018) 61–69

62

=+

μμ CsK Cs

l

s l

max,

, (1)

where μ, the specific growth rate is defined as:

= ⎛⎝

⎞⎠

μC

dCdt

1x

x

(2)

where, μ lmax, =Maximum specific cell growth rate, h−1

Ks,l = Substrate (lactose) saturation constant, g/LCs = Concentration of lactose, g/LCx=Concentration of L. casei, g/L

A double reciprocal plot of μ and Cs was plotted taking 1/μ and 1/Cs

as ordinate and abscissa respectively. The plot was found to be linearand the values of μ lmax, and Ks,l; i.e., the kinetic growth parameters wereobtained from the y-intercept and slope. Detailed calculations areshown in Section A of Supplementary.

3. Theoretical analysis

3.1. Mathematical model for probiotic microcapsules and beads suspendedin lactose-rich modified MRS medium and green coconut water: prediction ofinternal concentration profiles and release time

3.1.1. Schematic representation of microcapsules/beadsThe bead or microcapsule has been schematically represented in

Fig. 1.Although thorough washing of oil has been done, the external layer

of oil is present in case of microcapsules obtained via internal/externalgelation but not in case of beads synthesized via direct entrapment.

As shown in Fig. 1, a differential volume element, enclosed within rand r+Δr, has been considered. The following assumptions have beenmade:

1. The diffusion of lactose occurs from the bulk, into the interior of thecapsule and bead.

2. Lactose is the only limiting carbohydrate responsible for the growthof immobilized L. casei suspended in lactose-rich green coconutwater and modified MRS.

3. The mass transfer resistance due to oil layer is negligible for thetransport of lactose from the exterior to the internal core of themicrocapsules. This may be justified by the fact that the oil layer isthoroughly washed and hence its thickness is almost negligible.

4. The bulk concentration of lactose in the modified MRS medium andthe lactose-rich green coconut water remains constant over the

whole period of incubation. This is justified due to low diffusion andconsumption rates of lactose with respect to its total quantity ori-ginally present in the system and the same has been illustrated insubsequent sections.

Therefore, the mathematical formulations of the concentrationprofiles for beads prepared via different methods are same. By per-forming the lactose balance over the differential volume element fortime Δt:

− = ⎡

⎣⎢− ⎛

⎝∂∂

⎞⎠

+ ⎛⎝

∂∂

⎞⎠

+ ⎤

⎦⎥

++

πr ΔrCs πr ΔrCs D πr Cr

D πr Cr

πr Δrηr Δt

4 4 4 4

4

t Δt ts

r

s

r Δr

l

2 2 2 2

2

(3)

where, +Cs t Δt and Cs t are the concentrations of lactose at time t + Δtand t; +Cs r Δr and Cs r are respectively the values of lactose concentra-tion at radius r+Δr and r; D is the diffusivity of lactose in Ca-alginate, rlis intrinsic reaction rate and ƞ is the effectiveness factor (Macfarlaneet al., 1998).

Dividing both sides of Eq. (3) by π4 Δr Δt and limiting Δr→0 and Δt→0;

− =

⎛

⎝

⎜⎜⎜

− ⎞

⎠

⎟⎟⎟

+→

+

→

∂∂

+

∂∂( ) ( )r C r C

ΔtD

r r

Δrr ηrllim lim

Δt

s t Δt s t

Δr

Csr

r Δr

Cr

r

0

2 2

0

2 2

2

s

⎜ ⎟

∂∂

= ∂∂

⎛⎝

⎛⎝

∂∂

⎞⎠

⎞⎠

+t

r Cr

D r Cr

r ηr( )ss

l2 2 2

(4)

Dividing both sides of Eq. (4) by r2,

⎜ ⎟

∂∂

= ⎛⎝

∂∂

+ ∂∂

⎞⎠

+Ct

Dr

Cr

Cr

ηr2s s sl

2

2 (5)

Where,

= − ⎛⎝

⎞⎠

rY

dCdt

1l

x l

x

/ (6)

Where, Yx/l is the yield coefficient of biomass growth with respect tolactose and Cx denotes biomass concentration.

Since the growth of L. casei simultaneously on lactose followsMonod model,

Fig. 1. Probiotic L. casei embedded in alginate microcapsule/bead: Schematic representation.

S. Basu et al. Journal of Food Engineering 238 (2018) 61–69

63

=+

dCdt

μ C CK C

x l s x

s l s

max,

, (7)

Thus, Eq. (3) reduces to,

⎜ ⎟∂∂

= ⎡⎣⎢

∂∂

+ ∂∂

⎤⎦⎥

− ⎛⎝ +

⎞⎠

Ct

Dr

Cr

Cr

η μ C CK C

2Y

s s s l s x

s l s

2

2x/l

max,

, (8)

The initial and boundary conditions are as follows:

• I.C. (Initial Condition):

i) At t= 0Cs=00 < r < R

ii) At t= 0; Cx,i = 2.17 g/L; 0 < r < R (9)

• B.C. (Boundary Condition):i)At r= 0 = ≥t0; 0dC

drs

= = = ≥C C g L tii) At r R; 20 / ; 0s s b, (10)

Here, Cx,i and Cs,b denote initial biomass concentration and bulksubstrate concentration respectively. The various kinetic growth para-meters and constants are summarized in Table 1:

For different methods of encapsulation, the outside radius, i.e.; Rchanges. Samples of 10 beads/microcapsules synthesized by each im-mobilization method were chosen randomly from the total populationand observed under an optical microscope. The process was repeated 3times (total number of times the experiment was performed for eachimmobilization method). The configuration, mean size and standarddeviation of the total sample as observed, are reported in Table 2.

The above set of partial differential equations, coupled with theirB.C.’s and I.C.’s was solved using MATLAB R2010a to predict the con-centration profiles of biomass and lactose within the microcapsules(internal and external) and beads. The “release time” corresponding tothe maximum concentration of biomass in the immobilized matrix, hasbeen determined for all cases.

3.2. Concentration profiles of substrate and biomass in probioticmicrocapsules and beads using constant bulk substrate concentrationassumption: justification

Equations (7) and (8) were numerically solved using MATLABR2010a, coupled with their corresponding initial and boundary con-ditions (Equations (9) and (10)). For all simulations, the bulk con-centration of lactose in the abiotic environment, i.e., in the modifiedMRS medium and in green coconut water; has been assumed to beconstant. The justification for the assumption of constancy in bulkconcentration of substrate has been provided below:

3.2.1. For encapsulation via direct Ca-alginate entrapmentHere, we consider that substrate consumption is limited by diffusion

(as evident from substrate concentration profiles shown in Results andDiscussion section). Thus, mathematically;

⎜ ⎟⎛⎝

⎞⎠

= − −dC

dtk a C C( );s b

s b s i,

1 , ,(11)

where, Cs,b=Bulk concentration of substrate.Cs,i = Substrate concentration inside the cells.kl=Mass transfer coefficient of lactose in Ca–alginate.a= Specific interfacial area for mass transfer.

Solving the above equation using the required parameters (detailedcalculations are given in Section B of Supplementary), it is observedthat even after considering maximum diffusional driving force, the bulksubstrate concentration changes by only about 1.1% after 1 h, 2.3%after 2 h and 10.9% after 10 h. Therefore, our assumption of constantbulk concentration shall lead to incurrence of negligible amount oferror in our result.

3.2.2. For encapsulation via internal/external gelationFrom the concentration profiles obtained for internal and external

gelation (in Results and Discussion section); we may safely assume thatthe substrate concentration is uniform throughout the core of the cap-sules and equal to the bulk concentration. Therefore, substrate con-sumption, in this case, is limited by reaction. Making the necessarychanges to the generating equations (7) and (8) (detailed calculationsare shown in Section C of Supplementary), the generated results showthat the change in the bulk substrate concentration is negligible over arelatively prolonged period of time (0.25% over a period of 5 h), andhence, our assumption of unlimited substrate availability is justified.

3.3. Lactobacillus casei (2651 1951 RPK) cell concentration inimmobilized systems prior to bursting

Concentrations of cells in the internal aqueous core of Ca-alginatebeads and microcapsules (internal and external) at the “release time”have been calculated as follows:

The capsules (synthesized by any method) burst when their entirecore gets filled up by L. casei cells. Here, we assume no void space in thecell interior prior to bursting. Let the volume of an individual L. caseicell be Vc and that of a microcapsule synthesized by a particular methodbe Vcap. Hence, maximum number of cells that can be accommodated ina particular capsule is Vcap/Vc. Thereby, number concentration (Nc) ofcells in an individual capsule prior to bursting is given as:

= ⎛⎝

⎞⎠

=NcVcap

Vc Vcap Vcx 1 1 .

(12)

Now, Vc is unique and thereby, Nc is unique as well, devoid of anydependence on the method via which the capsules are synthesized.

Now, Dimensions of the bacterial strain (L. casei) (Banerjee et al.,2017b) are as follows:

Length= 2 μmDiameter= 0.5 μm

Table 1Summary of kinetic constants and initial/boundary conditions.

Sl. No. Parameter Description Value Source

1 D Diffusivity of lactose inCa-alginate

0.62×10−9 m2/s (Inoue, 1997)

2 Yx l Yield coefficient of L.casei growth withrespect to lactose

0.48 g/g (Doran, 1995)

3 μ lmax, Maximum specific cellgrowth rate

0.8336 hr−1 Experiment

4 Ks,l Substrate (lactose)saturation constant

2.64 g/L Experiment

5 Ƞ Effectiveness factor 1.0 Assumption6 Cx,i Initial biomass

concentration2.17 g/L Known value

7 Cs b, Bulk concentration ofsubstrate

20 g/L Known value

Table 2Outside radius of beads/capsules for different methods of bead formation/mi-croencapsulation.

Sl. No. Method Outside radius (R)

1 Ca–alginate entrapment 1.5 ± 0.02mm2 External gelation 14.7 ± 0.09 μm3 Internal gelation 6.0 ± 0.05 μm

S. Basu et al. Journal of Food Engineering 238 (2018) 61–69

64

Therefore, Vc= − −( )x (0.5x10 ) x 2x10π4

6 2 6

=3.92x10–19 m3

Nc= 1/Vc=2.55× 1018/m3

Approximately, weight of 109 cells is 1 mg (Van Dan-mieras et al.,1992).

Therefore, from the above-mentioned correlation, mass concentra-tion (Mc) of L. casei cells in a capsule (synthesized by any method) priorto bursting has been calculated as follows:

=

= × =

×( )M mg/m

2.55 10 mg/m 2.55g/mL

c2.55 10

103

9 3

189

Mc is more explicitly defined as the average mass concentration ofbiomass in the interior of the microcapsules/beads, which when at-tained, leads to bursting of the aforementioned microcapsules/beads.

4. Results and discussion

4.1. Mathematical modeling for the prediction of lactose and biomassconcentration profiles in probiotic microcapsules and beads suspended inlactose-rich modified MRS medium and green coconut water

4.1.1. Simulated concentration profiles for beads/microcapsules over theirincubation periods

The simulated concentration profiles of substrate and biomassconsidering their evolution over their incubation periods (ti); right upto their burst-release time have been shown in Fig. 2(a–b), Fig. 3(a–b)and Fig. 4(a–b) for Ca-alginate beads and microcapsules obtainedthrough external and internal gelation techniques respectively. Thetime-scales (900min for Fig. 2, 600min for Figs. 3 and 4) have beenchosen in the order of the burst-release times obtained using the threeencapsulation processes (868min, 587min and 574min for alginateentrapment, external and internal gelation respectively). The distinctcolored lines in Fig. 2(a) and (b) correspond to Cs and Cx at different r inthe respective figures. The bottom most line corresponds to r= 0 andthe uppermost line corresponds to r=R (external radius), with allother radii indicated by the intermediate lines. The arrows accom-panied by the letter ‘r’ in these figures show the direction in which theplotted radii ‘r’ increase.

3-D surface plots depicting radial concentration profiles of substrateconcentration for Ca-alginate beads are shown in Section D ofSupplementary.

4.1.2. Analysis of simulated profilesIn case of encapsulation via direct entrapment, the generation of

radial concentration profile of both substrate and biomass has beenobserved over the period of their incubation times. No such radialprofile of concentration of substrate is obtained for any of the micro-capsules. This may be due to relatively large diameter of the beadscompared to that of microcapsules. From the analysis of Fig. 2(a), it isevident that the saturation level of substrate concentration, i.e.; 20 g/L,corresponding to different radial positions, is attained around the40min range for Ca-alginate beads after which the curve dips mar-ginally. In case of microcapsules, the substrate concentration reachesthe saturation value of 20 g/L at each radial position (Figs. 3(a) andFig. 4(a)) almost instantaneously. On the other hand, steadily in-creasing trends in the time-concentration plot of biomass has beenobserved for all encapsulated systems (Figs. 2(b), 3(b) and 4(b)).However, unlike microcapsules, spatial gradient of biomass is observedfor the Ca-alginate beads, as depicted in Fig. 2(b). Figs. 3(b) andFig. 4(b) have been used to determine the times at which the burst-offbiomass concentration of 2.55 g/mL is attained in case of external andinternal gelation.

The time concentration profile of substrate for beads, depicted in

Fig. 2(a), clearly indicates a decreasing trend after reaching a maximumvalue at a particular incubation time of 40min at each radial position.This can be due to higher assimilation rate of substrate by the probioticcells in comparison to its rate of diffusion from the abiotic externalmedium. The biomass concentration, however, shows an increasingpattern over the entire time span. In case of microcapsules, while thesubstrate concentration remains saturated, increasing pattern of time-concentration plot of biomass is sustained (Figs. 3–4). As evident fromthe previous calculation, the maximum attainable biomass concentra-tion in all types of immobilized system is 2.55 g/mL. This value isachieved at 587 and 574min for the microcapsules produced throughexternal and internal gelation respectively.

From the analysis of Fig. 2(b), it is clear that beyond 450min, thevalues of biomass concentration at different radial positions at anyparticular time are different for Ca-alginate beads, the largest valuebeing at r=R and the smallest at r= 0. This is contrary to the natureobtained in case of internal/external microcapsules where biomassconcentration at no instant is a function of radial position (In fact, theindividual graphs shown in Figs. 3(b) and 4(b) represent Cx at differentradii, which merge into a single curve as the biomass concentrationprofile is same for all radial positions). Therefore, to calculate the‘burst–off’ time for the beads, the space–averaged concentration ofbiomass, Cx , at any particular time, has been used. Thus, the burst off

Fig. 2. (a): Evolution of substrate concentration (Cs) over time (t), for differentradii (r) for direct Ca–alginate entrapment. (b): Biomass concentration (Cx)profile for direct Ca–alginate entrapment.

S. Basu et al. Journal of Food Engineering 238 (2018) 61–69

65

time for beads has been defined as the time at which Cxreaches thesaturation value; 2.55 g/mL. Cx has been defined as follows:

∫= =

=

CC dr

Rxr

r R

x0

(13)

For this, using Fig. 2(b) and the output obtained from MATLAB, thefunctionalities of Cx on r have been determined at t= 500min,600min, 700min, 800min and 900min. Using the correlations, theCxat those values of time have been calculated using the above formula,as shown in Table 3 (The high values of standard deviation indicatelarge variations in biomass concentration within the alginate core).

The burst-release time has been determined corresponding toCx =2.55 g/mL via interpolation. This value has been determined to be868min and is in good agreement with the experimental value of880min.

The simulated values of the burst–release time (for all threemethods) have been compared with the experimental ones obtained forlactose rich modified MRS medium and the experimental values ob-tained using green coconut water as suspension medium, containing

initial lactose concentration of 20 g/L in Fig. 5 and good agreement hasbeen established indicating the validity of the model.

This mathematical model is expected to be applicable for the pre-diction of the behavior of encapsulated probiotics used for the pre-servation of food. Neutraceuticals or functional foods, food supplementsand other food ingredients are some of the forms of probiotic-basedfood that can have an important effect on the intestinal microorganisms(Parwal and Pareek, 2011). The delayed release of probiotics due to theimmobilization clearly enhances their availability for prolonged

Fig. 3. (a): Substrate concentration (Cs) profile (3-D surface plot) for externalgelation. (b): Biomass concentration (Cx) profile for external gelation.

Fig. 4. (a): Substrate concentration (Cs) profile (3-D surface plot) for internalgelation. (b): Biomass concentration (Cx) profile for internal gelation.

Table 3Average L. casei concentration in the interior of Ca-alginate beads as a functionof time.

Time(min)

Average biomass concentration in bead interior (Cx)(g/cm3)

Standard deviation(g/cm3)

500 0.23 0.22600 0.452 0.69700 0.818 2.01800 1.32 5.24900 3.91 12.89

S. Basu et al. Journal of Food Engineering 238 (2018) 61–69

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preservation period (Kawatra and Aiyappa, 2015). Since the bacter-iocin, caseicin in case of Lactobacillus casei having antimicrobial activityagainst pathogenic bacteria can diffuse out of the encapsulated en-vironment due to their small size, the shelf life of the preserved food isenhanced even when the probiotics are in immobilized form. After therelease of probiotics into the food matrix, their growth is sustained tillthe supply of nutrients is ensured. The survival of encapsulated pro-biotic organisms in another food matrix, viz carrot juice has been re-ported by Nazzaro et al. and Ying et al. during the studies on pre-servation at 4 °C (Nazzaro et al., 2009; Ying et al., 2013).

The mathematical model developed under this study is the first onefor immobilized probiotics suspended in food matrix and it is expectedto be successfully implemented for further designing of probiotic food.Some other factors that need to be considered while designing suchneutraceuticals include the requirement that the food contains the re-quired minimum viable probiotic count at the time of ingestion. Asrecommended by US FDA, the probiotic count in probiotic food shouldbe 106 CFUmL−1 at the least (De Prisco and Mauriello, 2016; Guarneret al., 2009).

It may also be noteworthy that although the encapsulated probioticsare released by bursting of beads/microspheres, however, this isbrought about in a controlled manner by limiting substrate availabilityto the bead/capsule interior. If cells are suspended in a free matrix, thisshall not be possible and unlimited substrate availability shall result inrapid, uncontrolled proliferation of the probiotics.

4.2. Experimental “burst-release time” of microorganisms from entrappedsystems of probiotics exposed to modified MRS medium and lactose-richgreen coconut water and validation of simulated analysis

The experimental values of “release time” of probiotic cells from theCa-alginate beads and different microcapsules exposed to modified MRSmedium and lactose-rich green coconut water are provided in Table 4.As is evident, burst-release time for beads and capsules vary with themethods through which they are formed. As per expectation, themaximum time taken is for Ca-alginate beads. This is due to the pre-sence of high diffusional resistance of substrate i.e. lactose and lowestsurface area to volume ratio. On the other hand, the microcapsulesproduced through internal gelation are ruptured first.

From the comparison of release time of probiotics from the

immobilized systems in modified MRS medium and lactose-rich greencoconut water, it is clear that the values obtained for modified MRSmedium are always slightly lower than those obtained for green co-conut water. This is due to the fact that although the probiotics aresupplied with same concentration of lactose (20 g/L) for both themedia, the essential micronutrients present in modified MRS are notavailable in green coconut water. Therefore, the growth rate of cellswithin the immobilized system suspended in modified MRS medium isexpected to be higher than that obtained in green coconut water.

Referring to Fig. 5, the absolute error between the simulated andexperimentally recorded burst-release times is 0.7%, 1.2% and 1.4% forinternal gelation, external gelation and alginate beads in MRS mediumrespectively. Therefore, the experimental and analytical values matchwithin the limits of experimental error. Hence, our model for gen-erating the simulated profiles and “burst-release times” is valid.

5. Model evaluation

The objective of the present study was to design a suitable modelthat can simulate the concentration profile of externally supplied sub-strate (lactose in this case) and biomass growth profile in the core ofalginate beads/microspheres within which a probiotic (L. casei in thiscase) was entrapped. This model not only helped to understand themechanism of reaction–diffusion by which the substrate permeates intothe alginate matrix but also predict the time after which the capsulesburst as a result of biomass proliferation within the beads/micro-spheres. This was validated vis-à-vis experimental results. This modelmay thereby be extended to simulate biomass growth and substratepermeation in case of other kinds of biomass encapsulated in a multi-tude of matrices and subject to a variety of nutrient media. In this study,

Fig. 5. Experimental and simulated burst-release times for beads and microcapsules in lactose-rich modified MRS medium and green coconut water. (For inter-pretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Table 4Experimentally obtained burst-release time of immobilized probiotic cells inlactose-rich modified MRS medium and green coconut water.

Immobilization method Experimental “burst-release time” (minutes)

Modified MRS Green coconut water

Internal gelation 570 ± 04 572 ± 04External gelation 580 ± 05 585 ± 03Ca-entrapment 880 ± 07 890 ± 05

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we assumed substrate availability as the only parameter governingbiomass growth. However, food matrices are subjected to varyingconditions of temperature, pH, mechanical stress etc. during theirprocessing/storage which may also affect biomass proliferation sig-nificantly. Other nutrients in the suspension matrix may also contributeto biomass growth. Future studies may incorporate these factors in theirresearch.

6. Conclusion

A deterministic, distributed mathematical model capable of pre-dicting the radial concentration profiles of concentrations of substrateand biomass within probiotic microcapsules/beads suspended in mod-ified MRS medium was developed for the first time. The burst-releasetimes of beads/microspheres were predicted and later, validated. Apopular, naturally-available rehydrant–green coconut water was alsoused as suspension medium for L. casei beads/globules and burst–re-lease times were calculated. The burst-release time for probiotics inlactose-rich coconut water is comparable with that obtained in case ofmodified MRS medium. It was established that microencapsulation/bead synthesis can help in obtaining a significant time–lag prior torelease of probiotics in the food matrix. This lag can enable the biomassto sustain thermal shocks, pH fluctuations etc. to which the food matrixis subjected to, during its processing and storage. Additionally, it canhelp prolong the shelf life of the product. It may also be noted that thisis not an overtly prolonged lag, which ensures the eventual release ofthe encapsulated biomaterials; thereby allowing their interaction withthe target. It may be concluded that application of probiotic micro-capsules in carbohydrate-rich liquid medium/drink will be beneficialfor sustained release of microorganisms, particularly due to elongationof time-lag prior to release. In addition, the realistic mathematicalmodel developed under this study shall be useful for the prediction ofburst-release time of similar formulations used for fortification of dif-ferent probiotic health drinks.

Conflicts of interest

There is no conflict of interest.

Acknowledgement

The second author (Debolina Banerjee) acknowledges the financialsupport offered by Council of Scientific and Industrial Research (CSIR),India by providing Senior Research Fellowship (File number: 9/96(0725)2K12-EMRI).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jfoodeng.2018.06.005.

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