https://doi.org/10.52973/rcfcv-e33296

Received: 24/07/2023 Accepted: 05/09/2023 Published: 13/10/2023

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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33296

ABSTRACT

Encapsulating materials preserve the viability of probiotics under

gastrointestinal conditions. The aim of the research was to evaluate

the protective effect of an encapsulating matrix, composed for the rst

time with three prebiotic materials to maintain the viability of a mixed

culture of spray–dried microencapsulated probiotics under simulated

gastrointestinal and prebiotic conditions. Microcapsules of four

formulations with better viability were then evaluated by inoculating

microencapsulated and free strains in MRS broth, adjusting three pH

values, bile salts, broth with and without carbohydrate (prebiotic test),

incubated at 36 ± 1°C / 24 h; then the percentage of post–treatment cell

survival was calculated. Showing that, formulation 1 presented higher

barrier protection with average counts: 7.31 log CFU·g

-1

lactobacilli and

7.75 log CFU·g

-1

(Saccharomyces boulardii) / 4 h (SGF), reaching 6.78 log

CFU·g

-1

in the four formulations (SIF) with a higher average survival rate

79.79% and 85.06% SGF and SIF, in vitro. On the other hand, the prebiotic

test maintained average counts of 9.40 log CFU·g

-1

(Lactobacillus spp.)

and 6.99 log CFU·g

-1

(S. boulardii) / 24 h. The protection exerted by the

microspheres under simulated gastrointestinal and prebiotic conditions

at therapeutic levels (≥ 10

CFU·mL

-1

) was demonstrated.

Key words: Gastrointestinal barrier protection; probiotic strains;

prebiotics; survival; microencapsulated strains

RESUMEN

Los materiales encapsulantes conservan la viabilidad de los probióticos

en condiciones gastrointestinales. El objetivo de la investigación fue

evaluar el efecto protector de una matriz encapsulante, compuesta por

primera vez con tres materiales prebióticos para mantener la viabilidad

de un cultivo mixto de probióticos microencapsulados por secado por

aspersión, bajo condiciones gastrointestinales y prebióticas simuladas.

Seguidamente, se evaluaron las microcápsulas de cuatro formulaciones

con mejor viabilidad, inoculando cepas microencapsuladas y libres en

caldo MRS, ajustando tres valores de pH, sales biliares, caldo con y sin

carbohidrato (prueba prebiótica), incubados a 36 ± 1 °C / 24 h; luego

se calculó el porcentaje de supervivencia celular postratamiento.

Demostrando que, la formulación 1 presentó mayor barrera de protección

con recuentos promedio: 7,31 log ufc·g

-1

lactobacilos y 7,75 log ufc·g

-1

(Saccharomyces boulardii) / 4 h (SGF), alcanzando 6,78 log ufc·g

-1

en las

cuatro formulaciones (SIF) con una mayor tasa de supervivencia promedio

79,79% y 85,06% SGF y SIF, in vitro. Por otra parte, la prueba prebiótica

mantuvo recuentos promedio de 9,40 log ufc·g

-1

(Lactobacillusspp.) y

6,99 log ufc·g

-1

(S. boulardii) / 24 h. Se demostró la protección ejercida

por las microesferas en condiciones gastrointestinales y prebióticas

simuladas a niveles terapéuticos (≥ 10

ufc·mL

-1

Palabras clave: Barrera gastrointestinal; protección; cepas

probióticas; prebióticos; supervivencia; cepas

microencapsuladas

Survival of a mixed culture of microencapsulated probiotic strains against

the gastrointestinal barrier in vitro

Supervivencia de un cultivo mixto de cepas probióticas microencapsuladas

frente a la barrera gastrointestinal in vitro

Luz Alba Caballero–Pérez

1,2

* , Rene Tejedor–Arias

, Elaysa Josena Salas–Osorio

University of Pamplona. GIBA Research Group. Pamplona, Norte de Santander, Colombia.

University of Havana, Pharmacy and Food Institute. Havana, Cuba.

Universidad de los Andes, Department of Biopathology. Mérida, Venezuela.

*Corresponding author: luzcaballero@unipamplona.edu.co

Microencapsulation improves probiotic survival in vitro gastrointestinal model / Caballero–Pérez et al. ____________________________

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INTRODUCTION

Probiotics are live microorganisms that when consumed in adequate

amounts colonize in the digestive tract, conferring a health benet

to the host, which is why today the food industry shows a growing

interest in the incorporation of probiotic microorganisms for the

elaboration of Functional Foods (FA), particularly in fermented dairy

products (yogurt, fermented milk and cheese) and non–fermented

products such as ice cream [1]. The medicinal or therapeutic ecacy

of these foods depends on the number of colony forming units per

mL or gram (CFU·mL

-1

or g) of viable probiotic microorganisms in

the product at the time of consumption. The minimum amount

recommended by the US FDA (Food and Drug Administration) and

the food industry in general was 10

CFU·mL

-1

[2].

According to Avila–Reyes et al. [3], the ability of probiotic

microorganisms to survive and develop in the host will directly

inuence their probiotic effects. Probiotic bacteria must be protected

from the adverse environment represented by the food matrix and

the gastrointestinal tract, in order to avoid a negative sensory

impact when they are incorporated into foods, as supplemented,

exposed to food conditions that could be adverse or favorable: pH,

humidity, temperature, oxygen concentration, water activity, nutrient

availability, presence of inhibitors, among others, which can affect

their viability and stability during storage and commercialization as

functional foods. Therefore, it is necessary to improve preservation

and protection conditions that guarantee the integrity of probiotics

when they are incorporated and processed in food matrices, as well

as to ensure their viability and activity when they are released in the

intestine where their action is required [3, 4].

The acid–tolerant capacity of bacteria is one of the common

characteristics among microorganisms of the Lactobacillus genus [5,

6]. Lactobacillus plantarum strains are considered a unique probiotic

because of their ability to resist acidic conditions by possessing

cellular mechanisms to maintain intracellular pH close to neutrality,

withstanding lower pH values than most other microorganisms [5]. On

the other hand, it was evaluated the viability of L. rhamnosus in relation

to the conditions of an acid medium, caused by fermentation, with the

resistance of the microorganism to pH close to 4.0, demonstrated

that this species produces lactic acid eciently through carbohydrate

intake, preferably using hexoses [7]. The yeast Saccharomyces

cerevisiae var. Boulardii, has been widely used as a preventive and

therapeutic agent in the treatment of diarrhea under the presentation

of lyophilized or heat–dried, the Word Gastrointestinal Organization

(WGO) recommends consuming 5 × 10

CFU of S. boulardii for certain

gastrointestinal disorders [8, 9, 10]. The ability of S. boulardii yeast

to grow over a wide pH range is known [8, 11].

One of the methods to prevent the decrease of cell load and/or

damage of probiotic bacteria is encapsulation. Microencapsulation

is a process by which bioactive materials are coated with other

protective materials or their mixtures as an alternative to maintain

high viability of microorganisms, protecting the core material from

environmental stress, such as oxygen, high acidity and gastric

conditions, and can be used to cross the gastric barrier with little

harmful effects. Among different microencapsulation techniques,

spray drying is commonly used because of advantages such as

low operating costs, high production rates, low moisture content

in the nal product, and possibility of industrial–scale application

[12], involves atomization of the feed solution in the hot air–drying

chamber, where water is evaporated from the atomized droplets to

form a dry powder [13]. Prebiotics dened as the food of probiotics

and generally represented by oligosaccharides and bers that are not

digestible by humans, are resistant to gastric acidity and digestion

by small intestine enzymes and provide a fermentable carbohydrate

for probiotic bacteria in the colon. High molecular weight β–glycan

glucose polymers (polysaccharides) are found naturally in the cell wall

of various living organisms such as bacteria, yeasts, fungi and plants

(cereals such as oats (Avena sativa) and barley (Hordeum vulgare)) [14].

Their combination can reinforce each other's effect, hence,

the synergistic combination of probiotics and prebiotics, termed

"symbiotics", which improves viable counts of lactobacilli and

bidobacteria compared to probiotic or prebiotic alone [9, 15]. The

addition of prebiotics in the encapsulation of probiotic microorganisms,

in what could be dened as a "co–encapsulation", can favor the viability

and ecacy of these benecial microorganisms in the gastrointestinal

tract. Some authors have reported that this co–encapsulation can

improve the functionality of the immobilized microorganism, generating

higher counts compared to encapsulation without prebiotic [16].

Several studies have been carried out on microencapsulation using

encapsulating materials such as: sodium alginate, calcium alginate,

chitosan, modied starch, maltodextrin, xantha gum, among others

[17, 18], achieving the preservation of probiotic microorganisms during

food storage and processing; However, they have the disadvantage

that all materials are porous to a greater or lesser degree, which

allows ion exchange directly affecting the pH inside the capsule

and consequently decreases the bioactivity of probiotics, reason

why the trend today is to use mixtures of encapsulating materials

to enhance and increase the viability of microorganisms improving

their protective properties against gastric and intestinal barrier

conditions [13, 19].

However, the survival of a mixture of three probiotic microorganisms

(two lactobacilli and one yeast) microencapsulated by spray drying

in a system composed of three prebiotic polymers as encapsulating

material subjected to in vitro gastric juice conditions has not been

evaluated. Therefore, the present research proposed to evaluate the

protective effect of a prebiotic encapsulating matrix on the survival of

the encapsulated microorganisms, using for the rst time a mixture

of sodium alginate, native cassava (Manihot esculenta) starch and

oat our (β–glycan) against gastrointestinal and prebiotic barriers

in vitro, as an innovative alternative.

MATERIALS AND METHODS

The work was carried out at: Nanotechnology Laboratory, University

of Pamplona, Laboratory of Applied Engineering and Bioprocesses

and Fermentations Berastegui headquarters, University of Cordoba,

Colombia. A pilot spray dryer, Vibrasec S.A©, model PSALAB 1.5,

stainless steel co–current ow, Medellín, Colombia, was used.

Encapsulating agents

The following were used: Native cassava starch. Starch Factory of

Sucre SAS, Innovayuca©; Sodium Alginate SQ 942. Trademark Cape

Crystal Brand©; Oatmeal. Original Ground Oats, Trademark Quakert ©.

Methods

The population was comprised of 28 mixtures of encapsulating

material obtained through the Design expert version 10 program and

supplemented with an approximate probiotic microorganism count

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of 1 × 10

CFU·mL

-1

. The sample was made up of four formulations

composed of sodium alginate, oat flour (β–glycan) and native

cassava starch. In order to dene the best formulation to be used in

microencapsulation, restrictions were dened: moisture content

(3 to 4%), viability (> 8 CFU·mL

-1

) and viscosity (100 to 250 mPa.s),

[3, 4, 20, 21]

The sample consisted of four formulations composed of sodium

alginate, native cassava starch and oat our (β–glycan) in different

percentages (%) respectively: formulation 1 (0.49 – 2.13 – 9.38);

formulation 2 (0.49 – 9.38 – 2.13); formulation 3 (0.62 –7.90 – 3.48);

formulation 4 (0.92 – 10 – 1.08).

The probiotic microorganisms evaluated were: Freeze–dried strains

of Saccharomyces boulardii, CNCMI – 745, Biocodex SAS©, using YGC

broth, Merck©, Sabouraud Dextrose Agar, Acumedia© brand for

their counts; Suspension strains of L. plantarum JCM1149, isolated

strain in the Biotechnology Laboratory; Freeze–dried strains of L.

rhamnosus GG, Merck©, there were used for counting broth and agar

MRS, Scharlau© brand.

The cell viability of free and microencapsulated probiotic

microorganisms (four selected treatments) subjected to Simulated

Gastric Fluid (SGF), Simulated Intestinal Fluid (SIF), and prebiotics,

was evaluated, selecting the treatment with the best resistance to

the three in vitro conditions.

Evaluation of the in vitro viability of microencapsulated probiotic

microorganisms to gastric and intestinal uid barrier conditions

The resistance of free and microencapsulated microorganisms

under the condition of SGF and SIF, [22, 23], with modications. To

represent SGF conditions, MRS broth was prepared, supplemented

with 1% pepsin by adjusting the pH to 1.0, 2.0 and 3.0 using

concentrated HCL [22], To prepare SIF, the amount of bile salts

necessary to reach a concentration of 0.1, 0.2, 0.3% (v/v) was

dissolved in MRS broth, adjusting the pH to 7.5.

The broths obtained were sterilized according to the manufacturer's

instructions. A 1:10 dilution was made from 1 g of the mixture of

microencapsulated probiotic strains and 1 mL of a mixture of strains

without microencapsulation (control) in 9 mL of peptonized water

homogenizing with constant agitation for 30 min. 100 µL of each

mixture was inoculated into 10 mL of SGF and SIF, respectively,

incubated at 36 ± 1 °C. Counts were performed by serial dilutions at

times 0, 1, 2, 2, 3, 4 h to assess cell viability [22]. Percent survival was

determined according to the following equation [3]:

(%)

log

Percentage of survival

CFUN

100

EC. (1)

Where: N

represents the total number of viable cells after the

treatments (SGF–SIF) and N

represents the initial number of viable

cells inoculated before the treatments (SGF–SIF).

Evaluation of the viability of microencapsulated microorganisms

submitted under in vitro prebiotic conditions

Based on the formulation of the MRS broth, model broths with and

without the main carbon source (sucrose) were prepared and 10 mL

tubes were prepared, which were inoculated with 100 µL of the 1:10

mixture obtained in the previous methodology and incubated at 36 ±

1°C. Counts of the SGF and SIF cultures were performed by means of

serial dilutions at times 0, 1, 2, 3, 4, and 24 h to assess the capacity

for cell multiplication. Additionally, the pH and the titratable acidity

were determined [3].

All the data were statistically analyzed using a factorial ANOVA, with

the viability of the probiotic strains as an independent variable with

a condence level of 95%. It was performing multiple comparisons

by Tukey test to establish where there were signicant statistical

differences between the four formulations and the viability of the

mixture of microencapsulated microorganisms.

RESULTS AND DISCUSSION

Evaluation of the in vitro viability of microencapsulated microorganisms

under gastric, intestinal and prebiotic barrier conditions

The results obtained in the different viability tests under gastric,

intestinal and in vitro prebiotic barrier conditions are presented below.

Viability of a mixed culture of microencapsulated probiotic strains

in a prebiotic matrix under Simulated Gastric Fluid (SGF) barrier

conditions in vitro

FIG. 1, shows how formulation 1 presents a higher barrier of protection

in the three microencapsulated strains subjected to pH (1.0, 2.0 and 3.0),

maintaining average counts of 7.31 log CFU·g

-1

for lactobacilli and 7.75 log

CFU·g

-1

for S. boulardii during 4 hours of exposure. While microcapsules

of formulations 2, 3 and 4 showed lower barrier protection; however,

they maintained counts above the recommended therapeutic level

(> 6 log CFU·g

-1

). At the same time, greater protection was observed

in the encapsulated lactobacilli with an average reduction of 1.82 log

CFU·g

-1

, and in the yeast a reduction of 2.05 log CFU·g

-1

; while the free

microorganisms showed an average reduction of 3.52 CFU·mL

-1

log,

demonstrating that the four formulations used with different mixtures

as wall material when forming the microcapsules, provide different

degrees of protection to the encapsulated strains of L. plantarum, L.

rhamnosus and S. boulardii strains.

Several researchers indicate that the production of symbiotic

microcapsules with the combination of wall matrix employing prebiotic

material (fruit–oligosaccharides (FOS), denatured whey protein isolate

DWPI) by spray drying method has potential applications in functional

food industry with good results [16, 24, 25].

As shown in TABLE I, formulation 1 composed of sodium alginate

(0.49%), native cassava starch (9.38%), oat our (β–glycan) (2.13%),

shows the highest in vitro survival rate among the four formulations,

maintaining the viability of the mixed culture of three microencapsulated

probiotic strains with an average of 79.79% during 4 h of exposure at pH

values 1.0, 2.0 and 3.0 in SGF, in vitro. On the other hand, formulations

2, 3 and 4 show an average survival rate of 47.030 and 49.10%, being

statistically different with respect to formulation 1 and the free

microorganism strains (P<0.05). On the other hand, formulations

2, 3 and 4 show an average survival rate of 47.030 and 49.10%,

being statistically different with respect to formulation 1 and the

free microorganism strains (P<0.05). It is observed that there are

signicant differences (P<0.05) in the average survival rates at the

three pH values evaluated (1.0, 2.0 and 3.0) being higher the average

survival rate at pH 3.0 with 63.726, while the type of probiotic strain

does not present signicant differences in the average survival rate

(P<0.05) indicating that the strains used have similar behavior against

the simulated gastric barrier.

FIGURE 1. Viability of mixed culture of three microencapsulated strains against the Simulated Gastric

Barrier (SG) at an exposure time of 4 h. Lactob: Lactobacillus

Microencapsulation improves probiotic survival in vitro gastrointestinal model / Caballero–Pérez et al. ____________________________

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cells at pH 2.0 with respect to those microencapsulated by spray

drying using various materials (whey protein, milk powder, alginate,

chitosan, inulin, among others) conferring greater protection against

SGF, in vitro [10, 13, 18, 25, 26].

Viability of three strains of microencapsulated probiotic microorganisms

in a prebiotic matrix under intestinal barrier conditions SIF in vitro

The tolerance of prebiotic microcapsules to the bile salt

environment is an important property [31], The viability results of

the three microencapsulated strains against the SIF intestinal barrier

in vitro at an exposure time of 4 h are presented below (FIG. 2).

The results in FIG. 2 show that the three encapsulating materials

used in the four formulations tolerate SIF barrier conditions, in vitro,

at bile concentrations (0.1, 0.2 and 0.3%). The free strains showed

a reduction of 1 log CFU·g

-1

, with lower microbial growth at the bile

concentration of 0.3% (7.09 log CFU·g

-1

); however, the average counts

were higher than the required therapeutic values. A decrease of 3.05

log CFU·g

-1

was observed after 4 h of exposure to barrier conditions

(SIF, in vitro), reaching a mean value of 6.78 log CFU·g

-1

in the four

formulations, probably due to the protective effect exerted by the wall

of the prebiotic microcapsules, results similar to those reported by

other authors who have demonstrated in vitro the protective capacity

of different materials against the SIF barrier [11, 13, 18, 25, 26].

It can be observed in TABLE II that the prebiotic microcapsules

of formulation 1 presented statistically signicant differences with

P<0.005, with the highest average survival rate (85.060%) with respect

to formulations 2, 3, 4 and the free probiotic microorganism strains,

showing signicant differences with P<0.005 in the three values of

the average counts of microorganisms against the percentages

TABLE I

Survival rate of three microencapsulated probiotic

strains in a prebiotic matrix versus SGF, in vitro

Dependent variable: Microorganism growth (CFU·mL

-1

)

Independent variables Categories Mean P value

3 63.726

0.000

2 58.131

1 51.229

Formulation

Free of microorganisms 65.526

0.000

Formulation 1 79.790

Formulation 2 47.030

Formulation 3 47.030

Formulation 4 49.100

Type of strain

Lactobacillus 58.241

0.552

S. boulardii 57.149

Averages that do not share a letter are signicantly different

The above demonstrates that the mixture of polymeric materials

used in the four formulations evaluated, offers a protective barrier

to gastric simulation conditions in vitro because the prebiotic and

encapsulating properties are enhanced by combining sodium alginate

with oat our (β–glycan) and native cassava starch, which provide

bre and polysaccharides of high resistance to the gastric barrier,

exerting greater protection on the mixed culture of encapsulated

probiotic strains [11, 12, 25, 26, 27, 28, 29, 30]. Results that coincide

with authors who determined a strong reduction of viability in free

FIGURE 2. Viability of mixed culture of three microencapsulated strains against the intestinal barrier (SIF)

at an exposure time of 4 h. Lactob: Lactobacillus

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of bile evaluated (0, 1, 0.2, and 0.3%) showing that the mixture

of encapsulating materials used in formulation 1 offered a better

protective barrier against SIF conditions, in vitro maintaining

the viability of the mixed culture of probiotic strains in the three

concentrations of bile after 4 hours of exposure for both lactobacilli

and yeast S. boulardii.

Likewise, the survival rate according to the type of probiotic strain

evaluated showed no signicant statistical differences with a P>0.05,

between Lactobacillus and S. boulardii yeast, demonstrating that the

probiotic strains used have the capacity to tolerate bile salts, being

able to develop their metabolic activities without being completely

inhibited during the 4 h of exposure to SIF, in vitro, results that

coincide with research that have evaluated the capacity of tolerance

to bile of different strains [17, 18].

The results were similar to those reported by other authors who

found a relatively high tolerance of S. boulardii to temperature, acid

pH and bile salts up to 0.3% (w/w) using whey protein as encapsulation

material, adding CaCl

or gum Arabic as optimal wall material, achieving

high viability of S. boulardii, showing that microcapsules produced

at higher drying temperature (125°C) showed higher resistance to

gastric solution compared to lower drying temperature (80°C) with

low resistance to the solution of gastric juice, demonstrating that

the combination of different encapsulant materials enhances the

protective capacity of microcapsules improving viability against

simulated gastric conditions. Furthermore, viability decreased with

increasing exposure to SIF, in vitro [23, 24, 25, 30].

In the same sense, the results of this research suggested that

the combination of prebiotic polymeric materials (sodium alginate,

oat our and native cassava starch) of formulation 1, improves the

survival of the mixed culture of probiotic strains against in vitro

gastrointestinal simulation conditions, maintaining the average

survival between 79.79 and 85.060%, demonstrating that this mixture

of encapsulating material improves its protective barrier properties,

results similar to those of other authors that suggest greater stability

of the microcapsules produced with prebiotics than those that used

only other types of material, showing higher initial counts, with

respect to free bacteria [20, 26, 27, 28].

TABLE II

Survival rate of three probiotic strains microencapsulated

in a prebiotic matrix against SIF barrier, in vitro

Dependent variable: Microorganism growth (CFU·mL

-1

)

Independent variables Categories Mean P value

Bile

Bile 0,1% 74.898

0.000

Bile 0,2% 70.031

Bile 0,3% 64.117

Formulation

Free of microorganisms 72.978

0.000

Formulation 1 85.060

Formulation 2 67.426

Formulation 3 56.441

Formulation 4 66.503

Type of strain

Lactobacillus 71.054

0.055

S. boulardii 68.310

Averages that do not share a letter are signicantly different

Microencapsulation improves probiotic survival in vitro gastrointestinal model / Caballero–Pérez et al. ____________________________

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Viability of three strains of microencapsulated probiotic

microorganisms in a prebiotic matrix with and without a carbon source

TABLE III show the results of the prebiotic test carried out on

the four selected formulations, using controls with and without a

carbon source to verify the prebiotic capacity of the encapsulating

matrix. It was observed that there were significant statistical

differences between the average counts of the four formulations

and the controls with and without carbon source with P<0.05, with

formulation 1 having the highest average count (9.0958 log CFU·g

-1

) in

relation to formulations 2, 3, and 4 and the controls (with and without

carbohydrates), being this growth lower in the mixture of free strains

without carbohydrates (6.6758 log CFU·mL

-1

) compared to those

exposed to MRS media with carbon source (sucrose), which may

be related to the use of the carbohydrates present in the prebiotic

microcapsules as a carbon source in the four formulations evaluated

by the three strains of encapsulated microorganisms.

where yeast activity decreased with increasing exposure time,

showing that yeast has a more demanding carbohydrate requirement

to reproduce and survive than Lactobacillus, since yeasts do not have a

metabolic system to fragment polysaccharides. [4, 9, 32], needing the

availability of sugars for growth; Another reason to explain this dilemma

is the proportion of lactic acid bacteria, LAB, which produce acids

derived from metabolism (fermentation), inducing acidic conditions

necessary for their preservation by using the carbon source that served

as a substrate for the mixed culture of probiotic strains, while yeast

may be unable to change the acidic conditions in such a short time.

Many authors indicate that the consequence of the antagonistic

effect, derived from the metabolites accumulated in the medium,

are produced by the fermenting bacteria, especially acids such as

propionate, acetate or lactate and their negative impact on the

growth rate of yeasts, all of which would indicate that when LAB

concentrations increase and these release greater quantities of

substances into the medium, a phase of cellular quiescence or

temporary halt to the multiplication of other organisms such as

yeasts will occur [9, 33, 34].

FIG. 3 shows that the acidity production increased with increasing

exposure time (24 h) of the microcapsules in MRS broth without

carbon source (negative control), corroborating that the three

materials (sodium alginate, oat our (β–glycan) and native cassava

starch) of the microcapsules, were used as carbon source in the four

formulations by the mixed culture of microencapsulated probiotic

strains, nding that there are no statistically signicant differences

with P>0.05 between the means of the acidity percentage values of

the four formulations.

While the control samples (with and without carbohydrate source

(sucrose) presented signicant differences with P<0.05, indicating

that the production of lactic acid and the reduction of pH values, as

well as the simultaneous depletion of carbon sources as a reduction of

growth microbial, they explain that LAB and yeast used the prebiotic

material the microcapsules wall (starches, bers (β–glycan)) as a

substrate to produce acids derived from metabolism (fermentation),

inducing acidic conditions necessary for their preservation and

maintaining their viability during the exposure time (24 h), behavior

similar to that reported by other authors who found that encapsulated

probiotic microorganisms use the wall material as an energy source,

preserving their viability during the exposure time [3, 19, 32].

It should be emphasized that recent studies have shown that β–

glycan from oats possesses great prebiotic potential that cannot

be digested by human digestive enzymes or absorbed in the upper

intestinal tract, while possessing the ability to provide a carbon

source to the intestinal microbiota within the distal intestinal tract

region [15, 16], therefore it can be deduced that the use of oat our in

combination with other prebiotic materials enhances their properties,

offering a feasible alternative to maintain the viability of a mixed

culture of probiotic strains.

TABLE III

Viability of mixture of probiotic microorganisms

with and without carbon source

Dependent variable: Microorganism growth (CFU·mL

-1

)

Independent variables Categories Mean P value

12 9.93228

0.002

0 9.42142

8 8.94561

4 8.83226

16 8.58331

20 7.91603

24 6.54270

Formulation

with carbohydrates 11.8969

0.000

Formulation 1 9.0958

Formulation 2 8.1944

Formulation 3 8.1908

Formulation 4 7.5238

without carbohydrates 6.6758

Type of strain

L. rhamnosus 9.664

0.000

L. plantarum 9.129

S. boulardii 6.994

Averages that do not share a letter are signicantly different

The above is related to the exposure time, where statistically signicant

differences with P<0.05 are observed from time 0 to 24 h, with a decrease

in counts as the exposure time to MRS broth with and without carbon

source increases due to the depletion of the carbon source. Similar

behavior has been reported by other authors, who indicated that the

encapsulated microorganisms use the wall material as an energy source,

maintaining their viability during exposure [3, 4, 19, 25, 29, 32] .

It should be added that there were statistically signicant differences

with P<0.05, between the mean counts of Lactobacillus (9.664 and 9.129

log CFU·mL

-1

) and S. boulardii yeast (6.994 logCFU·mL

-1

) of the four

formulations boulardii (6.994 log CFU·mL

-1

) of the four formulations,

FIGURE 3. Percentage of acidity produced in MRS broth with and without carbon source (sucrose), in vitro

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CONCLUSIONS

The mixture of polymeric material composed of sodium alginate,

oat our (β–glycan) and native cassava starch, exerts a protective

barrier in gastrointestinal conditions in vitro, being an innovative

alternative to improve the survival of a mixture of strains of probiotic

microorganisms microencapsulated by spray drying, maintaining

therapeutic levels (≥10

CFU·g

-1

The best protective effect in the prebiotic microcapsules was in

formulation 1 composed of sodium alginate (0.49%), native cassava

starch (9.38%), oat our (β–glycan) (2.13%), showing a higher survival

rate of the mixed culture of microencapsulated probiotic strains in

the in vitro gastrointestinal and prebiotic barrier conditions.

The prebiotic microcapsule wall material in all four formulations,

composed of sodium alginate, oat our (β–glycan) and native cassava

starch serve as a carbon source maintaining the viability of a mixed

culture of microencapsulated probiotic strains.

ACKNOWLEDGMENT

Nanotechnology Laboratory of the University of Pamplona for their

support and collaboration in providing the facilities, equipment and

materials required for the development of the project.

Conicts of interest

All the authors state that there is no signicant nancial conict

of interest or any other type of interest that could arise in the results

or interpretation of this article.

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