SUMMARY

Initially, the antimicrobial susceptibility of L. plantarum and E. coli O157: H7 was

measured, where both were susceptible to cefotaxin, and ciprofloxacin. Also, the inhibitory

effect of L. plantarum and its supernatant against E. coli O157: H7 was evaluated. The

highest inhibition showed an 8mm halo. Additionally, by means of HPLC-DAD peptides

and amino acids were determined in the supernatant of the two bacteria; the peptide

corresponded to the sequence VAL-TIR-VAL, tyrosine being the predominant amino acid

in the two bacteria. Subsequently, in order to evaluate the growth of L. plantarum, its

fermentation kinetics was performed, presenting an exponential phase at 12:00 h with a

growth of 27.39 Ln CFU / 150 μl. Additionally, its growth was evaluated at two

temperatures (38 ° C and 45 ° C), its highest growth was at 45 ° C with 3.0x1012 CFU /

150 μl. Finally,in microencapsulated L. plantarum was evaluated: viability, efficiency of

microencapsulation, physical properties and structural characterization by scanning electron

microscopy (SEM). Relative humidity and water activity (Aw) were measured at 37 days of

storage, its relative humidity was 7.79% and Aw 0.400, efficiency was 46%, solubility

96%, wettability 1:56 minutes and its viability 58% . The effect of microencapsulation on

the probiotic viability of L. plantarum was evaluated under simulated gastrointestinal

conditions of pH 3.0, bile salts at 0.3% and 1% and 0.5% bovine bile at pH 5.5 per 24:

00h , reaching significant growths of 3.0x109UFC / 150 μl at pH 5.5 in bovine bile at 0.5%.

Key words: Microencapsulation, Escherichia coli O157: H7, Lactobacillus plantarum,

probiotic, spray drying.

 ABSTRACT

The effect of microencapsulation on the probiotic viability of Lactobacillus plantarum was

evaluated under simulated gastrointestinal conditions in Escherichia coli O157: H7. The

lactic strain was subjected to the following conditions: pH 3.0, for 24:00 hours; bile salts at

0.3% y 1% and bovine bile at 0.5%, with a pH of 6.5 in the last two tests for a period of

24:00 hours. As a result, significant growths were obtained such as 6.4x109CFU / 150 μl in

bovine bile. In addition, in the fermentation kinetics, it was found that the exponential

phase was at 12:00 hours with a growth of 27.39 Ln CFU / 150 μl. Furthermore, its growth

was evaluated against the temperature levels (38 ° C and 45 ° C) where the highest growth

was recorded at 45 ° C. The HPLC-DAD was used to determine the peptides and amino

acids in the environment in both bacteria, as a result, was obtained a VAL-TIR-VAL

sequence, Being the tyrosine the predominant amino acid in both bacteria. Antimicrobial

susceptibility testing was performed where the two bacteria were susceptible to cefotaxime,

and ciprofloxacin. Also, the inhibitory effect of L. plantarum and its supernatant against E.

coli O157: H7 was evaluated, where the greatest inhibition was presented with an 8mm

halo. Finally, viability, efficiency of microencapsulation, physical properties and structural

characterization were evaluated by Scanning Electron Microscopy. The relative humidity

and water activity (Aw) was measured after 37 days of storage, with the following results:

Humidity 7.79%, Aw 0.400, efficiency 46%, solubility 96%, wettability 1:56 minutes and

viability 58%.

Key words: Microencapsulation, Escherichia coli O157: H7, Lactobacillus plantarum,

probiotic, spray drying.

 INTRODUCTION

In the world, every year, thousands of people get sick due to the bacterium Escherichia coli

of a pathogenic serotype, with diarrheic symptoms and quite serious complications for the

host, the different outbreaks in the world have caused E. coli O157: H7 to be considered as

a public health problem (1). In order to control the effect of this type of bacteria and avoid

the use of antibiotics, there are investigations that have proposed probiotics as inhibitors of

pathogenic bacteria. In this sense, probiotics of the genus Lactobacillus have been

associated with several beneficial effects on human and animal health. Studies have shown

the bactericidal action of L. plantarum against E coli and emphasize that its action is due to

the reduction of pH, production of organic acids, production of biocines and good capacity

of adhesion to the intestinal mucosa that allows it to compete for space (2,3). On the other

hand, for a probiotic bacterium to manifest its beneficial effects, it must survive the passage

through the gastrointestinal tract and resist the different environmental and technological

conditions, therefore the protection of probiotic bacteria of the genus Lactobacillus is very

striking for an application in multiple food systems (4,5). In this sense, a technique to

improve viability is microencapsulation because it forms protective microcapsules with

elements that protect and serve as prebiotics around bacteria, protecting them during their

passage through the gastrointestinal tract and against environmental conditions (3). Thus,

the objective of this investigation was: to evaluate the effect of microencapsulation on the

probiotic viability of Lactobacillus plantarum under gastrointestinal conditions and

inhibition on Escherichia coli O157: H7.

 MATERIALS AND METHODS

The research was carried out in the laboratory of the Fise-Probiotec research group and in

the specialized laboratories of the University of Nariño, located in the Municipality of

Pasto, Department of Nariño, Colombia.

 Lactobacillus plantarum ATCC 8014 and Escherichia coli O157: H7 ATCC 43888® were

used. The reconstitution of each strain was carried out according to the manufacturer's

instructions and its preservation was realized by means of a peal in solid and liquid medium

every 5 and 8 days.

To obtain the culture of L. plantarum, an aliquot of the strain was deposited in an

Erlenmeyer flask with 40 ml of MRS broth and incubated for 24:00 h at 37 ° C;

subsequently, 4 ml of the incubation were taken and deposited in 40 ml of MRS broth, and

incubated under equal conditions.

The inoculum was adjusted by incubating 40 ml of MRS (lactic) or BHI (pathogenic) broth

during 24:00 h. Then, a 4 ml fraction was taken to be deposited in 40 ml of MRS or BHI

broth and incubated during 24:00 h Also, 10 ml of inoculum was taken and finally placed in

90 ml of MRS or BHI broth (6). At the end of the incubation, 1 ml of the inoculum was

added to 9 ml of peptonated water and the spectrophotometry (625 nm) was read in the

Genesys 10S UV-VIS Spectronic spectrophotometer, in cases where the population was

higher than the established one, sterile broth was added (6)

Inhibition against pathogens and comparison with antibiotics. The susceptibility of L.

plantarum and E. coli O157: H7 to the antibiotics cefotaxime (CTX 30 μg), penicillin (P 10

IU), gentamicin (GN 10 μg) dicloxacillin (DCX 1 μg), ciprofloxacin (CIP 5 μg) and
cefalotina (KF 30 μg) was evaluated. using the methodology of Kirby-Bauer et al (7). The

inhibition of the supernatant of L. plantarum on E. coli O157: H7 was also determined by

the methodology of Tagg and McGiven (8) with different concentrations. In order to

determine susceptibility of the pathogenic strain, inhibitory haloes greater than 2 mm were

taken into account, which are considered as indications of susceptibility (9).

Fermentation kinetics. The kinetic parameters of L. plantarum were evaluated in

commercial MRS medium. An Erlenmeyer with an adjusted inoculum was taken, incubated

during 24:00 h, samples and measurements were taken every 3:00 h to determine viable

plaque microorganism count (UFC / 150 μl), consumed sugar, protein produced, pH and

lactic acid. To determine the count of viable microorganisms in plaque, the Lanara

methodology was used (10). To determine the pH, a digital potentiometer (Jenco®

VisionPlus) was used. To determine the sugar consumption, different glucose

concentrations were prepared to create a standard curve using the values obtained from the

samples observed at an optical density of 525 nm (8). In the determination of protein

production, a calibration curve was made by bovine serum albumin and absorbance was

determined in a spectrophotometer at 625 nm, taking into account the methodology

proposed by modified Lowry (11). The determination of lactic acid production was carried

out by titration of sodium hydroxides (12).

The production of the biomass was evaluated in terms of time-growth rate during the

fermentation process through the method proposed by Crueger and Crueger (6). The

fermentation calculations for the kinetics were those defined by Crueger and Crueger (6)

and Rodríguez et al (13). A sample of supernatant of L. plantarum and E. coli O157: H7

was taken and by HPLC-DAD the content of peptides and amino acids was determined,
where for its analysis 1.0 ml of sample was taken through syringe filters of PVDF of 0.25

μm. Subsequently, HCl was added and centrifuged x 15:00 minutes at 9000 rpm. Samples

were taken to dryness with nitrogen stream and derivatized. The identification was made

using an amino acid standard in 0.1N HCl (Pickering, neutral amino acids) analyzed under

the same operating conditions developed in the Chromatography Laboratory of the

University of Nariño, using the following equipment: Liquid Chromatograph HPLC Waters

- Binary Pump 1525, column C18 300Á (Jupiter, Phenomeme x150 mm x 4.6 mm), PDA

detector 2998 to 214 and 280nm Scan (200-350nm) and Rheodyne Loop Injector 20μl.

The fermentation calculations for the kinetics are those defined by Crueger and Crueger (6),

Rodríguez et al (13) and Gamazo et al (14). The calculation of the specific speed of growth

(v max), time of duplication and time of generation and maximum harvest was carried out

according to the established by Gamazo et al modified (14), was calculated from the slope

of the product produced by graphing Ln x (UFC / 150 μl) vs time (hours), until the

logarithmic phase.

y = mx + b

Where m is the slope, thus v is max

The time of cellular duplication (td) was determined taking into account the following

equation:

td = Ln2 / (v max)

The time it takes to double the population or generation time (g) was calculated by the

formula:
 g = Ln 2 / vmax

The maximum harvest was calculated by the following expression:

M = M1-M0

The expression M1 belongs to the bacterial growth in the stationary phase and M 0 is the

growth at the beginning of the fermentation kinetics expressed in Ln UFC.

Resistance to different temperature levels. According to the methodology of Cai et al

(15) the growth of L. plantarum was evaluated at different temperatures (37 ° C and 45 ° C)

planted in MRS broth by 24:00 h. Subsequently, viability tests were performed on MRS

agar for 12 h at 37 ° C and 45 ° C. Petri dishes with viabilities of 30 and 300 CFU / 150μl

and dilution values equal to or greater than 107 CFU / ml were selected.

Microencapsulation of Lactobacillus plantarum by the Spray Drying Technique. The

bacterial biomass was obtained according to what was described according to Crueger and

Crueger (6). According to the methodology of Rodríguez et al (16) modified, a suspension

of 20% w / v (60 g of Maltodextrin and 60 g of Inulin in 280 ml of adjusted bacterial

inoculum) was performed in a ratio of 1: 1 w / w. The Bilon 6000s® Spray Dryer Drying

Equipment was used. The microencapsulated material was stored in dark plastic containers

at room temperature (20 ± 2 ° C).

Survival and Stability Study of Encapsulated Lactobacillus plantarum. To evaluate the

effect of microencapsulation on the viability of L. plantarum, the number of viable cells

was determined before and after spray drying after 37 days after being stored at 20 ± 2 ° C

(16). The following, were used as stability criteria: the viability of the lactic acid bacteria
(BAL) evaluated during storage (> 108 CFU / g), physical properties (Water Activity,

Relative Humidity, Solubility and Wetness), and Structural Characterization (Morphology

and size of the microcapsules), in addition, to the efficiency of the microencapsulation

according to that described by Gonzales et al (4). The size of the microcapsules was made

by means of scanning electron microscopy (SEM) in a scanning electron microscope FEG

(Field Emission Gun) QUANTA 650 FEG, for analysis the samples were sent to the

Microscopy and Microanalysis Center, Bogotá, Colombia. The samples were placed in

metal stubs with carbon adhesive tape and coated with gold in a Quorum 150ES coating

equipment and the respective images were taken.

Exposure of Lactobacillus plantarum microencapsulation to simulated

gastrointestinal conditions. The growth of microencapsulated L. plantarum was evaluated

at pH 3.0 and at concentrations of 0.3 and 1% of bovine bile salts, and 0.5 and 0.3% of

bovine bile at a pH of 5.5. (17) To that end, 2 g of the microencapsulation were added in 18

ml of MRS broth mixed with the percentage of bile and bile salts adjusted to the

corresponding pH, then incubated at 37ºC for 48:00 h for the release and establishment of

the microencapsulated material. The viability test was performed under the methodology of

Cai et al (15). It could be deduced that the bacteria were resistant to gastrointestinal

conditions based on the verification of the viability of the microencapsulated probiotic

subjected to the different concentrations of pH, bile and bile salts (17).

Statistical analysis. To establish the effect of the MRS medium on the L. plantarum

variable, an analysis of measures repeated over time was performed, using the procedure

PROC MIXED of the statistical package of SAS taking into account a level of significance

of 5% (p less 0.05).
Yijk = μ + αi + τk + (ατ) ik + eijk

Yijk = Value of the measured response for time k in subject j assigned to treatment i.

μ = General average.

αi + τk + (ατ) ik = Mean for the treatment i at time k, associated with the fixed effects

treatment (αi), time (τk) and the interaction treatment by time (ατ) ik.

eijk = Random error associated with subject j assigned in treatment i for time k.

To model the variables sugar, protein, acidity and pH, 3 polynomial models were used;

linear, quadratic and total cubic; through the coefficient of determination (r2) and the mean

square of the error, the best fit was selected. The mathematical models are placed below:

y_ij = 〖β_0 + β〗 _1 (Xi) + ε_ij

y_ij = 〖β_0 + β〗 _1 (Xi) + β_2 〖(Xi)〗 ^ 2 + ε_ij

y_ij = 〖β_0 + β〗 _1 (Xi) + β_2 〖(Xi)〗 ^ 2 + β_3 〖(Xi)〗 ^ 3 + ε_ij

y_ij: Ln UFC / 150 μl

β_0 to β_3: These are the regression coefficients

Xi: The time of sampling.

ε_ij: Error
 RESULTS AND DISCUSSION

Inhibition Against Pathogens and Comparison with Antibiotics. The results of the

antimicrobial susceptibility test in Lactobacillus plantarum and Escherichia coli O157: H7

are shown in Table 1 and Figure 1. The results of the In vitro test of lactic bacteria on

pathogenic bacteria are found in Table 2.

The lactic and pathogenic bacteria showed sensitivity to Cefotaxin and Ciprofloxacin.

Cefotaxin is widely used to treat respiratory, urinary, enteric and central nervous system

infections (18) and ciprofloxacin in human medicine, is used for urinary infections and

other locations, but also used indiscriminately and inadequately (19). Now, it can be

observed that the growth of L. plantarum as well as the pathogenic bacterium is inhibited.

Therefore, it is evidence where the use of antibiotics to eliminate pathogenic bacteria can

affect the survival of beneficial bacteria for the organism, generating long term imbalances

between beneficial and pathogenic bacteria. Regarding the resistance of pathogenic bacteria

to penicillin and dicloxacillin, its use is considered to control enteric diseases; generally

they are not used for this purpose (20). However, it is necessary to bear in mind that the

indiscriminate use of antibiotics for the treatment of infections in humans and animals has

caused resistance, subsequently these drugs are used without any previous analysis to

determine the specific antibiotic to be implemented, causing a problem to global level

which presumes great difficulties in the antibiotic treatment when it is required (21).

Regarding what was obtained in this study, susceptibility tests of antibiotics on L.

plantarum reached similar inhibition diameters with ciprofloxacin and higher with

gentamicin with diameters of 31 mm and 29 mm respectively (22). Another study reports

an inhibitory halo of the antimicrobial to the lactic bacteria of 30 mm for ciprofloxacin and
17 mm for gentamicin (23). The previous results coincide with other studies that report E.

coli against gentamicin and ciprofloxacin presenting a sensitivity profile with diameters of

22 mm and 42 mm respectively (3).

In relation to the above, Gutierrez et al (24) and Vegas et al (25) reported the inhibition of

lactic bacteria on E. coli. In this sense, the presence of peptides, may be related to their

bactericidal activity that inhibits the presence of pathogenic bacteria (26). Therefore,

greater inhibition halos with the use of the supernatant than with the impregnated bacteria

were obtained, since the bactericidal substances with inhibitory capacity are used when

using the supernatant. Inhibitory reports with the supernatant of L. plantarum showed halos

of 2.5 mm against E. coli, also mention that the main biocine produced by L. plantarum is

plantaricin, this compound has been shown to reduce the presence of bacteria present in the

environment where it is (3).

Fermentation Kinetics. The results obtained for percentage of lactic acid, pH,

microorganism count, protein production and sugar consumption, its comparison with Ln

CFU / 150μl can be found in figure 2. An exponential phase was found at 12:00 h (time 5),

with values of 4.44 for pH, 0.29% for lactic acid, 4.9 mg / L for proteins, 6.88 mg / L in

sugars and a growth of 27.39 Ln CFU / 150 μL, The kinetic data can be observed in table 3.

The statistical data of the effect of biomass Ln (CFU / 150μl) on the time of the culture

medium Lactobacillus plantarum are in table 4 and indicate significant statistical

differences (p <0.05) between time 1 and time 9 versus time 5. Linear and quadratic

regressions of pH variation, lactic acid% production, sugar consumption and protein

production are shown in table 5 and as a result it was obtained that both in pH, consumption

of sugars and production of protein there was an inverse correlation with the variable time,
and in terms of the production of% lactic acid has a direct correlation that is, every three

hours increases 0.10% lactic acid.

In the identification of peptides from the supernatant of the pathogenic and lactic bacteria, 4

peaks were detected. The sample presented the retention time similar to the peak of the

calibration standard corresponding to the peptide of composition VAL-TIR-VAL with a

concentration of 0.48 mg / ml. The chromatograms for L. plantarum and E. coli O157: H7

are found in Figure 3 and their amino acid composition in Table 7.

The results demonstrate the capacity of L. plantarum to colonize the digestive system

during 12:00 h of its exponential phase, since there is a maximum bacterial growth and that

they coincide with other studies where they mention a duration of growth of L. plantarum

48 : 00 h so from that time begins the death phase (27). In this perspective, as the bacteria

grows the lactic acid increases and the pH decreases. The main objective of lactic acid

bacteria is to cause an increase in acidity due to the production of organic acids (28), this

decrease in pH has an effect of inhibiting pathogenic or altering microorganisms in the

product in the case of the food industry, this is because there are few bacteria that are able

to grow at the pH levels reached by the action of the BAL. In addition, some lactic bacteria

are capable of producing bacteriocins, such as plantaricin in the case of L. plantarum. On

the other hand, the effectiveness in cell duplication and the number of generations per hour

indicates that its behavior is optimal since it serves to obtain commercial inocula and

probiotics due to its efficiency and therefore it will reduce costs (29), adding that, With this

level of growth lactic bacteria will have the ability to compete with pathogenic bacteria,

benefiting the host, reducing bacterial translocation and intestinal inflammation.

Other studies report that the logarithmic phase of L. plantarum was reached at 16:00 h with

cellular duplication times of 84.50 minutes (23). On the contrary, other authors coincide

with what was obtained in this study with a logarithmic phase at 12:00 h (3). The above,

indicates that the lactic strain worked has more efficiency when duplicated and can reach its

inhibitory effect faster. However, its death phase can reach faster, since it consumes more

quickly the nutrients present in the medium.

For bacteria it is important the presence of proteins or nitrogen compounds that favor their

metabolism and therefore their growth and development. Subsequently, this characteristic

favors industrial processes, since the BAL play a very important role during the

fermentation of milk, mainly in the fractionation of proteins to peptides and free amino

acids, originating bioactive peptides, which have immunomodulating, anticancer and

hypocholesterolemic effects (30) Bioactive peptides are defined as amino acid compounds

with inhibitory activity or bacteriocins (31). Amino acid chains have also been reported for

L. plantarum and E. coli from VAL-TIR-VAL (23, 3).

Within this perspective, the important bacteriocins in L. plantarum are the plantaricins. The

production of bacteriocins by bacteria is linked to bacterial growth (32); that is to say that

as the bacteria grows in mass, the greater their production of bacteriocins. However, when

their growth decreases or reaches a stationary phase the bacteriocin decays in production.

Plantaricin is a potion complex that consists of two different peptides that ensure a better

antimicrobial action. It also adds that the mechanism of action of plantaricin against

pathogenic bacteria is through electrostatic bonds with negatively charged phospholipids of

the cell membrane, then they are inserted into the membrane and the monomers of the

bacteriocin form protein aggregates that form the pore that promotes the release of ions

such as potassium and magnesium, motive power comes out of protons, ATP and amino

acids, as there is no active transport and movement afterwards of the cell, there is no energy

and therefore there is cell death (33).

Resistance to Different Temperature Levels. The obtained results are in Table 8. In the

genus Lactobacillus the optimum range of growth temperature is between 23 ° C-50 ° C

below the temperature, a latency state (34). Some authors highlight that the resistance of the

BAL to temperatures of up to 50 ° C, gives them the ability to survive body temperature,

preserve in the dormant phase at room temperature and survive technological processes in

the production of balanced feed and microencapsulated by spray drying medium where the

temperature rises to 300 ° C (6,35,36). Data obtained in one study show growths of 3x1012

CFU / 150 μl at a temperature of 45 ° C for L. plantarum (23).

Survival and Stability Study of Encapsulated Lactobacillus plantarum. Humidity was

measured 37 days after storage of the microencapsulated L. plantarum obtaining a

percentage of 7.79%. The water activity (Aw) is the amount of free water in a sample that

can be used by microorganisms or other destructive agents (17). This variable was verified

at 37 days of storage and was obtained 0.400 of Aw. The solubility that was obtained in the

sample was 96%. The wettability showed that the microencapsulated to disappear under the

surface of the water took 1:56 minutes. The efficiency of the microencapsulation was 46%

and its viability 58%. The structural results of microencapsulation can be seen in figure 4

and 5. Its morphology describes a simple sphere and in some cases irregular where the

coating or the membrane cannot be observed. (37).

The relative humidity percentage of microencapsulated L. plantarum is an important

variable, which can influence the conservation of live microorganisms (38). The humidity

values should be between 3.5 and 4% for the encapsulated material to remain stable during

storage (17). Since when the moisture content is high, microencapsulation becomes prone

to deterioration (39). It is important to note that despite the fact that the value exceeds the

optimum for the microencapsulated material. An investigation reports humidity percentages

of 6.37% and 7.03% for L. casei and L. rhamnosus microencapsulated by the spray drying

technique using the same microencapsulating materials (38). Therefore, the relative

humidity is similar to what was observed in the present study and does not necessarily

translate into the detriment of the microencapsulated material, as this was reflected in its

significant viability and growth over time. At the same time, there was no evidence of

contamination by foreign agents such as bacteria, fungi, among others, in the

microencapsulated material. However, relative humidity can affect the conservation of

microencapsulation (40 and 38). Since the lower the percentage of moisture, the better it

will be to preserve it. Additionally, it is important to analyze the microencapsulation

technique since, taking into account the values reported in other studies (38), the technique

used for microencapsulation can affect the moisture present in the final product.

Aw is an external agent that constitutes an adverse effect on probiotics, and that can be

reduced with the use of microencapsulation (38). A value of Aw that benefits the stability

of the microencapsulation should be close to 0.3 (38). The values of Aw obtained in the

microencapsulation of L. plantarum can be considered acceptable since they do not

represent values of high water activity, which can benefit the viability of the

microencapsulation. Values of Aw above 0.60 are susceptible to contamination with

bacteria, molds and yeasts (41). This means that the microencapsulation is in good

condition after 37 days of storage. The microencapsulation of probiotics has been verified

in L. casei and L. rhamnosus finding Aw values of 0.32 and 0.38 respectively, with the

same microencapsulating materials and the same microencapsulation technique (38), values

that are not far from obtained in this study. Therefore, microencapsulated L. plantarum is

among the optimal ranges to be properly conserved.

Different studies have reported solubility values for microencapsulated probiotics by means

of the lyophilization method with the same encapsulating materials obtaining solubilities of

98% (42), close to that obtained in this study. The high solubility is closely linked to the

chemical and structural characteristics, related to the number of free sugars and the degree

of polymerization (GP) (43). However, the rehydration properties of encapsulated powders,

such as solubility, can affect the viability of microorganisms as a critical step in their

recovery after drying, because cells that have undergone a possible sublethal injury may not

be able to to repair that damage if they rehydrate in inappropriate conditions (43).

The wetting of the particles is the process that controls the reconstitution and the speed of

cellular recovery (17). The wettability time obtained from the sample of L. plantarum

microencapsulated with inulin and maltodextrin is relatively low compared with the values

obtained in a study for bacteria of the genus Lactobacillus microencapsulated with inulin as

a prebiotic material and maltodextrin as a wall material with the same method of

encapsulation (L. casei 6.33 min; L. rhamnosus 4.52 min) (38). Montes emphasizes that a

slow rehydration given in controlled scenarios demonstrates better viability and maximizes

cell recovery compared with immediate rehydration (38). Therefore, short wettability times

such as those obtained can influence the viability and recovery of probiotics. The low
wettability time may be due to the humidity value of the microencapsulation, since this

percentage of the powders contribute to the wetting, because the liquid penetrates the pores

with greater ease depending on the degree of humidity (44).

The viability in this study was 58%. Other studies in microencapsulated L. plantarum

showed a lower result than that obtained in another study (38), since it reports viability

percentages of 84% and 96% in L. rhamnosus and L. casei respectively. In L. plantarum

efficiencies of 93% have been reported (38). According to the above, the viability of

microorganisms is this study goes over half, although there are significant growths it is

important to analyze other techniques or other microencapsulating materials that ensure the

viability of lactic bacteria.

Regarding efficiency with 46%, it indicates that microencapsulation recovery was poor

compared to other studies where they report efficiencies with the spray drying technique of

96-100%. (42) The efficiency of controlled release mainly depends on the composition and

structure of the wall, but also of the operating conditions during the production and use of

these particles (temperature, pH, pressure, humidity) so that the microencapsulation

production and its efficiency can be altered. (42)

The size reported in this study was 15.18 - 35.68μm. Another study, achieved sizes of

6.33μm and 6.86μm for L. casei and L. rhamnosus (38) values not very far from the lower

range obtained in the investigation. There are reports of 53.99μm microencapsulation using

other microencapsulating materials (43). The size of L. plantarum without

microencapsulation has a report of 1.2 μm long by 1.0 μm wide (45). On the other hand,

when large microcapsule sizes are reported, there is a greater possibility of contact with
external agents and as a consequence greater deterioration in storage (38). The

microencapsulation processes form capsules whose size ranges from 5 to 300 μm (37). In

addition, sizes between 10-100μm are considered fine powders (44). Therefore, the

microencapsulation of L. plantarum for being of small particle will exhibit less

deterioration during storage. According to other authors the ideal particle size should be

between 15-100μm, at a value lower than 15μm there may not be adequate protection for

the microencapsulated bacteria and on the contrary if it is greater than 100μm it is

susceptible to being attacked by external agents such as bacteria, fungi, among others (46).

The above indicates that the size of the particle is correct to carry out its protective

function.

Exposure of Lactobacillus plantarum microencapsulation to simulated

gastrointestinal conditions. The results of the present test can be found in Table 9.

In the evaluation of the microencapsulated lactic acid strain subjected to gastrointestinal

conditions, some authors recognize an important probiotic characteristic of lactic acid

bacteria, such as their capacity to resist acidic media, because they release organic acids

since they need rich media to grow them. sugars to favor acidification and survival (29).

This indicates that the microencapsulated bacterium is capable of surviving the first barrier

that is the stomach (pH 3.0), which has the function of eliminating potentially pathogenic

bacteria by submitting them to fairly acidic gastric juices, therefore the microencapsulated

bacteria can exert its inhibitory effect by surviving acidic media. On the other hand, one

study reports good growth of L. plantarum without microencapsulation under bile salts and

bile reaching growth values of 1x1013 CFU / 150 μl at 4% bile salts and 6x108UFC / 150μl

at 0.5% bovine bile (23 ), in the present study, greater growth was obtained in bovine bile
with respect to bile salts than reported in the previous study in question, which suggests that

the growth of the bacteria once microencapsulated may affect the viability, but not

significantly. Rodríguez et al (44) studied the effect of microencapsulation on a probiotic

where they obtained positive results in terms of viability with respect to storage time,

further highlighting that a viability of 21 days may exist. However, microencapsulation is

not only aimed at ensuring its passage through TGI, but also keeping the viable bacteria

when stored at room temperature, since the microencapsulating materials, in addition to

having a protective wall effect, also have prebiotic effects.

CONCLUSION

Microencapsulation by the spray-dried technique favored the probiotic action of L.

plantarum. That is to say, that I not only help in terms of its viability at the intestinal level

but also its conservation at room temperature for a longer time. In addition, this bacterium

demonstrated its probiotic action against a pathogenic bacterium which is a public health

problem, important as an infectious agent associated with food poisoning, affecting mainly

children and the elderly. This, thanks to the presence of bacteriocins and their modification

of the environment where they are making difficult the survival of bacteria that support acid

media. Its kinetic behavior demonstrated that it is a bacterium that can be used at an

industrial level thanks to its rapid growth which would reduce the costs when performing

bacterial inocula with these lactic bacteria. It is important to carry out In vivo studies since

In vitro showed important characteristics as probiotic strain.

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TABLES AND GRAPHS

Table 1. Susceptibility test of microbial agents against Lactobacillus plantarum and

Escherichia coli O157: H7

L. plantarum Halo L. plantarum E. coli Sensitivity

Antibiotic
(mm) Sensitivity m Halo (mm) E. coli
CTX 30 µg 30 Sa 32 Scd
P 10 IU 17 Rab - Rc
GN 10 µg 15 Sab 17 Scd
DCX 1µg - Rab - Rc
CIP 5 µg 19 Sab 40 Sc
KF 30 µg 13 Rab 18 Scd
S: sensitive I: intermediate R: resistant. Cefotaxin (CTX); Penicillin (P); Gentamicin (GN);
Dicloxacillin (DCX); Ciprofloxacin (CIP); Cefalotin (KF) G.

a: Charteirs et al (53) b: Jurado et al (28) c: Bernal et al (54) d: Cavalieri et al (55)

Table 2. Halos inhibition of L. plantarum on E. coli O157: H7 in supernatant and
impregnated agar disks

Concentration and
Method Condition measurement of the halo
75 µl 85 µl 95 µl
Filtered pH 6.0 2mm 2mm -
Filtered pH 6.0 y 80°C 5mm 2mm 3mm
Discs Method Pads Unfiltered pH 6.0 2mm 6mm 2mm
Unfiltered pH 6.0 y 80°C 5mm 5mm 7mm
80 µl 100 µl 120 µl
Filtered pH 6.0 3mm 5mm 4mm
Diffusion in plastic cylinder Filtered pH 6.0 y 80°C 5mm 4mm 2mm
Unfiltered pH 6.0 - 3mm 2mm
Unfiltered pH 6.0 y 80°C 2mm 3mm 2mm
Filtered pH 6.0 2mm 4mm 2mm
Diffusion in double layer Filtered pH 6.0 y 80°C 4mm 3mm 8mm
plastic cylinder Unfiltered pH 6.0 - 3mm 4mm
Unfiltered pH 6.0 y 80°C 2mm 2mm -
50 µl 75 µl 100 µl
Impregnated agar discs
Müeller Hinton Agar 6mm 5mm 4mm
Table 3. Kinetic data of the bacterial growth of Lactobacillus plantarum in the MRS
medium.

Strain Medium
L. plantarum MRS
Latency phase UFC/150µl <3
Specific speed of growth (µ h-1) 3,105
End of the logarithmic phase (hours) 12:00
Cell duplication time (minutes) 13,39
Number of generations per hour 4,48
Maximum harvest Ln CFU/150µl 9.91
Cell increment / end of the logarithmic phase 3,0E+14
UFC/150µl
Total cellular increase 1,2E+08
% of sugar consumed at the end of logarithmic 17,53
phase
% Total of sugars consumed 38,60
% of protein consumed at the end logarithmic 28,25
phase
% of total protein production 57,31
R2 end of logarithmic phase 0,9193

Table 4. Effect of biomass Ln (CFU / 150μl) on the time of the culture medium

Lactobacillus plantarum.

Lactobacillus plantarum
Time 1 2 3 4 5 6 7 8 9
Ln UFC/ 21,0bc 21,2bc 21,7b 27,3ab 30,3a 29,1ab 23,3ab 18,4c 18,4c
150µL c
Similar letters indicate that there is no significant statistical difference, different letters
indicate significant statistical differences p <0.05

Table 5. Regressions of pH, % lactic acid, consumption of sugars and production of

protein over time of L. plantarum

variable Regression Pending Intercept r2

 pH Quadratic -0.136(t) 0.003(t2) 5.572 0,978
% lactic acid Linear 0.10 -0.171 0.945
Sugar consumption Quadratic -0.677(t) -0.020(t2) 77.991 0.961
Proteins production Quadratic -0.101(t) -0.506(t2) 512.167 0.910

Table 6. Identification of amino acids L. plantarum and E. coli O157: H7.

E. coli O157:H7 L. plantarum

No Aminoacids Relative amount % Relative amount %
1 Aspartic acid 4,5 5,0
2 Serine 8,9 9,0
3 Arginine 8,0 8,7
4 Tyrosine 50,7 51,6
5 Valine 10 15,7
6 Proline 2,0

Table 7. Viability of Lactobacillus plantarum in MRS medium at temperatures of 37°C

and 45 ° C (CFU / 150 μl)

L. Plantarum
Dilution Factor UFC/ 150 µl
37°C 45°C
105 3.0 X 107 3.0 X 107
106 3.0 X 108 3.0 X 108
107 4.2 X 109 4.2 X 109
108 - -
109 - -
1010 - 3.0 X 1012

GRAPHIC 1. Inhibition halo, cefotaxime on L. plantarum and ciprofloxacin on E. coli

O157: H7
GRAPHIC 2. Growth of L. plantarum CFU / 150 μl in MRS medium
GRAPHIC 3. Peptide chromatogram of the supernatant of the pathogenic strain E.
coli O157: H7 and lactic L. plantarum.

GRAPHIC 4. Microphotography by scanning electron microscopy (SEM) capsules of

L. plantarum morphology.
GRAPHIC 4. Microphotography by scanning electron microscopy (SEM) capsules of
L. plantarum morphology.