In vitro

evaluation of the probiotic potential of microorganisms isolated from functional

commercial

fermented milks

João Victor Ferreira Campos

, Mariana Oliveira Silva

, Ana Carolina Alves Vieira

, Luiza Mota de Faria

, Gustavo Lucas

Costa Valente

, Priscila Mara Rodarte Lima e Pieroni

, Marcelo Resende de Souza

, Jacques

Robert Nicoli8, Leonardo Borges

Acurcio9*

DOI: https://doi.org/10.35699/2447-6218.2021.35380

Abstract

Fermented milk is one of the carriers of probiotics, which is a Greek derived term meaning “pro-life”. Probiotics are live

microorganisms that, when administered in adequate and daily amounts, confer benefits to host’s health. In this work, four in

vitro tests were performed with six lactobacilli samples from commercial fermented milks: sensitivity test to gastric pH,

sensitivity test to intestinal bile salts, antagonism test against pathogens and susceptibility test to antimicrobials. The tested

samples were resistant to gastric pH (2.0) and some suffered growth inhibition in the sensitivity tests to bile salts (0.3%), in

two different methods, reducing growth in about 45% or 1 Log10 reduction. All

pathogenic bacteria tested (Escherichia coli,

Salmonella enterica serovar Typhimurium and Staphylococcus aureus) were

antagonized by the supernatant (in MRS) of

Lactobacillus spp. isolated from commercial fermented milks as well as in spot on the lawn test. Regarding antimicrobial

susceptibility, samples presented a diverse profile, with prevalence of sensitivity to antimicrobials of clinical relevance, although

there was high resistance profile regarding cephalosporins. In view of the in vitro tests performed, samples showed a satisfactory

probiotic potential, as expected. However, some samples showed a slightly superior performance, such as Lactobacillus casei

Shirota from Yakult, L. casei Defensis from Actimel and L. paracasei ST11 from Chamyto.

Keywords:

Antagonism. Lactobacillus. Resistance to gastrointestinal transit.

Avaliação

in vitro

do potencial probiótico de micro-organismos isolados de leites

fermentados

funcionais comerciais

1Centro Universitário de Formiga (UNIFOR-MG) – Formiga, MG.

https://orcid.org/0000-0003-0902-0000

2Universidade Federal de Minas Gerais (UFMG) – Belo Horizonte, MG.

https://orcid.org/0000-0002-0537-1666

3Universidade Federal de Minas Gerais (UFMG) – Belo Horizonte, MG.

https://orcid.org/0000-0002-9284-8054

4Centro Universitário de Formiga (UNIFOR-MG) – Formiga, MG.

http://orcid.org/0000-0001-9726-056X

5Universidade Federal de Minas Gerais (UFMG) – Belo Horizonte, MG.

http://orcid.org/0000-0001-8256-0383

6Centro Universitário de Formiga (UNIFOR-MG) – Formiga, MG.

http://orcid.org/0000-0001-6621-6777

7Universidade Federal de Minas Gerais (UFMG) – Belo Horizonte, MG.

http://orcid.org/0000-0001-7071-7304

8Universidade Federal de Minas Gerais (UFMG) – Belo Horizonte, MG.

https://orcid.org/0000-0003-2390-2608

9Centro Universitário de Formiga (UNIFOR-MG) – Formiga, MG.

https://orcid.org/0000-0002-2981-5479

Corresponding author. leoacurcio@uniformg.edu.br.

Recebido para publicação em 27 de julho de 2021. Aceito para publicação em 28 de setembro de 2021

e-ISSN: 2447-6218 /

ISSN: 2447-6218. Atribuição CC BY.

CADERNO DE CIÊNCIAS AGRÁRIAS

Agrarian Sciences Journal

Campos, J. V. F. et al.

Resumo

O leite fermentado é um dos veiculadores dos probióticos, que é um termo derivado do grego que significa “a favor da vida”.

Probióticos são microrganismos vivos, que quando administrados em quantidades adequadas e diárias, conferem benefícios à

saúde do hospedeiro. Neste trabalho foram realizados quatro testes in vitro com amostras de lactobacilos de seis leites

fermentados comerciais: teste de sensibilidade ao pH gástrico, teste de sensibilidade aos sais biliares intestinais, teste de

antagonismo contra agentes patogênicos e teste de suscetibilidade aos antimicrobianos. As amostras testadas foram resistentes

ao pH gástrico (2.0) e algumas sofreram inibição do crescimento nos testes de sensibilidade aos sais biliares (0,3%), em dois

tipos de teste in vitro diferentes, com redução de aproximadamente 45% no crescimento ou cerca da 1 Log10. Todas as bactérias

patogênicas testadas (Escherichia coli, Salmonella enterica sorovar Typhimurium e Staphylococcus aureus) foram

antagonizadas pelo sobrenadante (em MRS) dos Lactobacillus spp. isolados de leites fermentados comerciais assim como em

teste de antagonismo to tipo spot on the lawn. Quanto à suscetibilidade aos antimicrobianos, as amostras apresentaram perfil

diversificado com predominância para a sensi- bilidade a antimicrobianos de importância clínica, apesar do elevado perfil de

resistência às cefalosporinas observado. Frente aos testes in vitro realizados, as amostras apresentaram potencial probiótico

satisfatório.

Palavras-chaves:

Antagonismo. Lactobacillus. Resistência ao trato gastrointestinal.

Introduction

Concerned about a healthier life, more and more

people have been looking for better life habits, associating

physical exercise with a balanced diet (Berti el al., 2017).

Knowing this, food industry, aware of consumer prefe-

rences, has raised its concern over healthy and functional

food

and beverage market (Granato et al., 2010). In this

context,

fermented milk or fermented dairy product stand

out as an

attractive vehicle for probiotic cultures (Barat; Ozcan, 2017).

The objective of this work was to evaluate the in

vitro fuctional features of microorganisms isolated from

commercial functional fermented milks. For this, in vitro

sensitivity of microorganisms to gastric juice and bile salts

was evaluated, as well as antagonistic activity of

microorganisms against pathogenic bacteria and sus-

ceptibility to antimicrobials.

Material and Methods

Commercial functional fermented milks found in

retail chain of Minas Gerais were evaluated, namely:

Actimel from Danone (Danone, Paris, France), Batavinho

from Batavo (Batavo, Carambeí, Brazil), Chamyto from

Nestlé (Nestlé, Vevey, Switzerland), Itambé from Itambé

(Itambé Alimentos, Belo Horizonte, Brazil), Vigor from

Vigor (Vigor Alimentos, São Paulo, Brazil), and Yakult

from Yakult (Yakult, Tokyo, Japan), from which single

species of probiotic Lactobacillus sp. were isolated after

count, which is described below. All products were bought

within expiry date, under refrigeration (<7°C) and were

maintained at this temperature until experimentation.

The International Dairy Federation defines fer-

mented dairy product as a dairy product prepared from

skimmed or non-skimmed milk with specific cultures

(Panesar, 2011). The main bacteria used in its produc- tion

are usually lactobacilli, being responsible for the sensory

characteristics of the product and the reduction of its pH.

Addition of probiotic microorganisms in the

product

reinforces its functional properties by increasing its nutritional

and therapeutic value, also resulting in the

improvement of its

sensory characteristics (Sharifi-Rad et al., 2020).

Probiotics have its’ beneficial properties only

when administered in adequate amounts and in viable

conditions (FAO/WHO, 2002). So, a minimum viable

quantity for probiotics was established, ranging from 108

and 109 Colony Forming Units (CFU) in the daily

recommendation of the product (ready for consumption).

addition, for a product to be classified as a probiotic, used

culture must have resistance to bile salts and gas- tric acidity

confirmed by laboratory tests, according to Brazilian

legislation (Brasil, 2018). Resistance to gastric and intestinal

injuries is of great importante in probiotic selection, since a

minimum of 106 CFU is required for a probiotic sample to

exert is possible beneficial effects. If not resistant, bacterial

count may reduce drastically and probiotic effect will not

occur (Vinderola; Reinhemer, 2003).

Count of viable microorganisms was carried out

from six different samples of commercial fermented milk.

Initially, a serial dilution of up to 10-5 of 0.1mL of

each

sample was made in 0.9mL of sterile peptone saline

(0.9%

NaCl, 0.1% peptone). Afterwards, 0.1mL of the

solution

obtained from each dilution was plated with the

aid of the

Drigalski’s loop. Solution was then distributed over the

surface of petri dishes containing MRS (de Man Rogosa and

Sharpe - MRS) agar (Difco, Detroit, USA). Plates were

incubated aerobically for 24 hours at 37°C. Colonies with

different morphotypes were subjected to Gram staining and

catalase tests, in order to confirm it as lactobacilli.

Antibiogram was performed according to the

technique of antimicrobial susceptibility by the principle

Cad. Ciênc. Agrá., v. 13, p. 01–09, DOI: https://doi.org/10.35699/2447-6218.2021.35380

In vitro evaluation of the probiotic potential of microorganisms isolated from functional commercial fermented milks

of drug diffusion in disc. Discs with pre-defined drug

concentrations were used and the diameters of the halos

formed by inhibition were measured following the one

proposed by Charteris et al. (1998a).

of placing 3.5ml of soft agar (0.75% BactoAgar, Difco, in

broth of Brain Heart Infusion - BHI, or MRS, Difco) with

revealing bacteria. The methodology used in this test was

adapted from Acurcio et al. (2014).

Each microorganism was transferred to a test tube

containing 3.5mL of 0.9% saline until the concentration

equivalent to the 0.5 McFarland scale (corresponding to an

estimated population of 108 CFU/mL). Culture was then

spread on a pizza-type Petri dish (14cm diameter) with a

sterile swab until the entire surface containing MRS agar

(Difco) was covered. Then, in an equidistant manner, 10

disks (Laborclin, Pinhais, Brasil) containing

antimicrobials

from different chemical groups were distri- buted. Drugs

belonging to the following chemical groups

were used:

Revealing bacteria were the following pathogenic

microorganisms: Enterococcus faecalis ATCC 19433, Esche-

richia coli ATCC 25922, Salmonella Typhimurium ATTCC

14028, Shigella flexneri ATCC 12022 and Staphylococcus

aureus ATCC 29213. 10μL of the microorganisms that had been

previously incubated were transferred to semi-solid

agar and

poured onto MRS agar plates containing the previously

described spots.

Finally, plates were incubated at 37°C for 48

hours, under aerobiosis. Inhibition halos were read using a

digimatic caliper (Mitutoyo). This test was performed in

duplicate - the same revealing microorganism applied to two

plates with the same isolated microorganism tested (spot) -

with two replicates.

Cell wall synthesis inhibitors, with bactericidal

action, such as beta-lactams: penicillin (PEN, 10U), am-

picillin (AMP, 10μg). Glycopeptides: such as vancomycin (VA,

30μg). Third generation cephalosporins: ceftriaxone

(CRO,

30μg), cefoxitin (CFO, 30μg). Protein synthesis

inhibitors,

with bacteriostatic action, such as tetracyclines: tetracycline

(TE, 30μg); as well as aminoglycosides: gen- tamicin (GEN,

10μg), chloramphenicol (CLO, 30μg) and streptomycin (EST,

30μg). Cell multiplication inhibitors,

with bactericidal action,

such as the third generation quinolones: ciprofloxacin (CIP,

5μg).

Second method: in vitro antagonism methodolo-

by Acurcio et al. (2017) was used, with adaptations.

Initially, microorganisms isolated from the six samples of

fermented milk were grown in MRS broth (Difco) at a

temperature of 37°C for 24 hours, under aerobic condi-

tions. Then, 1mL of each probiotic sample was pipetted into

three different microtubes. Microtubes were centri- fuged at

5.000g for 5 minutes and, supernatants were

obtained. The

pH of each supernatant was measured and

its microfiltration

was carried out, in order to ensure the exclusive action of

culture supernatants.

After the distribution of disks, Petri dishes were

incubated in aerobiosis at 37°C for 48 hours. For quality

control of disks containing antimicrobials, a sample of

Escherichia coli ATCC 25922 was used. Finally, diame-

ters of the inhibition halos were read using a Mitutoyo

digimatic caliper (Mitutoyo, Suzano, Brasil). The test was

performed in duplicate with two repetitions. Cha-

racterization of the antimicrobial susceptibility profiles of

the evaluated samples was carried out according to

Charteris et al. (1998a).

The reference microorganisms (Escherichia coli

ATCC 25922, Salmonella enterica serovar Typhimurium

ATCC 14028 and Staphylococcus aureus ATCC 29213)

were inoculated in BHI broth (Brain Heart Infusion) and

subsequently incubated at 37°C for 24 hours. Afterwards, in

three microtubes of each pathogenic sample, 4% (v/v)

solutions were prepared containing 960μl of BHI broth

(Difco) added, individually with 40μl of one of the pa-

thogenic microorganisms. Then, 100μL of the probiotic

supernatant was transferred to an ELISA plate with 96

wells

and another 100μL of the solutions described above.

The plate

was then incubated in a spectrophotometer

(Microplate

Spectrophotometer System SpectraMax 340

- Molecular Devices, San Jose, USA) for 12 hours at 37°C.

The

OD 620nm reading at every 30 minutes determined the

absorbance of the culture.

Before the next test was carried out, lactic acid

bacteria isolated from each fermented milk was grown in

5mL

of MRS broth (Difco) and incubated for 48 hours at

37°C

under aerobic conditions. Then, in vitro antagonism test

against pathogenic microorganisms was performed in

two

different ways, each technique being used for every six

samples.

First method: five microliters of each microor-

ganism culture were placed on the center of the surface of a

Petri dish, containing MRS agar (Difco), which was

incubated under aerobiosis at 37°C for 48 hours. After 48

hours, the plates were removed from the incubation

chambers with the spots in the center of the plate grown.

Chloroform was placed on the plate covers and left for 30

minutes under ultraviolet (UV) light to perform its action.

With this process, the microorganisms that grew in the spots

were eliminated, allowing the evaluation of

supposed

inhibitory substances produced by the bacteria

and released in

the culture medium. Next step consisted

To calculate the percentage of growth inhibition,

the

GraphPad Prism 6.01 (GraphPad, San Diego, USA)

program was used. The formula (1-AT / CT) x 100, with AT

corresponding to the area under the growth curve of the

revealing pathogenic microorganism in probiotic

supernatant and CT correspond to the area under the growth

curve of the control pathogenic microorganism, was adapted

form Andrade et al. (2014). The control

consisted of adding

the solutions of pathogenic samples in

pure MRS broth

(Difco), in which there was no previous

Cad. Ciênc. Agrá., v. 13, p. 01–09, DOI: https://doi.org/10.35699/2447-6218.2021.35380

Campos, J. V. F. et al.

growth of any probiotic microorganism. The tests were

performed in triplicate, with two repetitions.

First technique (intestinal bile salts): To measure

sensitivity to bile salts, the technique described by Santos

et al.

(2016) was adapted. The isolated microorganisms were

activated in MRS broth (Difco) and incubated at 37°C

under aerobiosis for 48 hours. Previously to the assay, the

microorganisms were plated at serial decimal dilutions at 10-

4 and 10-6 on MRS agar (Difco) in order to define the control

growth of each of the lactobacilli samples (incubation at

37°C, for 48 hours, aerobically).

Subsequently, 1ml of each

sample was centrifuged in 2mL

microtubes at 5.000g for 5

minutes. The supernatant formed was discarded and the

samples exposed to 1ml of MRS broth solution (Difco)

containing 0.3% (w/v) of bile salts (Oxgall, Difco) for six

hours. At the end of the six hours, plating was done from

decimal dilutions serialized at 10-4 and 10-6 on MRS agar

(Difco) in order to assess the viability of the cells. Finally,

plates were incubated at 37°C for 48 hours in aerobiosis. The

results

found were compared with the plating results made

prior to the test to observe the behavior of the microorganisms

studied in an artificial intestinal environment.

Evaluation of the sensitivity or resistance of mi-

croorganisms to gastric pH and intestinal bile salts were also

carried out in two different ways.

First technique (gastric simulation): To measure

the

sensitivity to gastric pH, the technique described by Santos

et al. (2016) was primarily used. The isolated

microorganisms were activated in MRS broth (Difco) and

subsequently incubated for 48 hours at 37°C under aerobic

conditions. In order to define the control growth of each of the

lactobacilli samples, the microorganisms were plated prior to

the test in the serial dilutions at 10-4 and 10-6 on MRS agar

(Difco) (incubation at 37°C, for 48 hours, aerobically).

Later, 1ml of each sample was centrifuged in 2ml

microtubes at 5.000g for 5 minutes.

The obtained supernatant

was discarded and the samples

were exposed to 1 ml of

0.9% saline solution (pH 2.0,

0.3 w/v of pepsin), for one hour. After the determined time,

a plating based on serial dilutions at 10-4 and 10-6 on MRS

agar (Difco) was performed, with the purpose of evaluating

the viability of the cells. Finally, the plates were incubated at

37°C for 48 hours, aerobically. The results obtained were

compared with the results of the plating done before the test

to observe the behavior of the microorganisms studied in an

artificial stomach en- vironment.

Second technique (intestinal bile salts): To assess the

resistance of microorganisms isolated from fermented milk to

bile salts, the tests were based on the methodolo-

gy described

by Acurcio et al. (2017). First, the isolated

microorganisms

were activated in MRS broth (Difco) and

incubated in an

aerobic condition at 37°C for 24 hours. After incubation, the

activated microorganisms were

placed in microtubes at a 4%

(v/v) dilution in MRS broth (Difco). Then, 100μL were

transferred to one well on the 96-well ELISA plate containing

100μl of pure MRS broth

(Difco) and another 100μL were

transferred to another well on the same plate containing

MRS broth (Difco) with 0.6% (w/v) of bile salts (Oxgall). To

determine the

absorbance of the culture, the reading of OD

620nm was

performed, every 30 minutes for 12 hours.

Second technique (gastric simulation): Adap-

tations were performed over methodolgy proposed by

Acurcio et al. (2014). First, the isolated microorganisms were

activated in MRS broth (Difco) and incubated for 24 hours

at 37°C, under aerobic conditions. After incu- bation, 1mL

of each sample of the activated microorga- nisms was added

in two different microtubes that were centrifuged at 5.000g

for 5 minutes. The supernatants

formed after centrifugation

were discarded and the pellet

obtained was suspended in

saline with pH 2.0 and 0.3 w/v of pepsin (gastric pH) or 7.0

(control). After that, the samples were incubated at 37°C for

one hour and centrifuged again under the same conditions

mentioned above. After centrifugation, the pellet was

suspended in MRS (Difco). 2% (v/v) inoculum was also

prepared in MRS broth (Difco). The sample was then

distributed in a 96-well ELISA plate, with 200μl of each

sample after

passage through pH 2.0 (gastric pH) and pH 7.0

(control).

In the end, the inoculum of samples was 2% and

the

concentration of bile salts 0.3% in the wells, simu- lating, in

vitro, intestinal environment. To calculate the percentage of

growth inhibition, the program GraphPad

Prism 6.01

(GraphPad Software) was used, which carried

out the

determination by the formula (1-SB / CT) x 100,

with SB

corresponding to the area under the growth curve

of the control

bacteria treated with bile salts and CT, the control. The tests

were performed in triplicate, with two repetitions.

The plate was incubated in a spectrophotome- ter

(Microplate Spectrophotometer System SpectraMax 340 -

Molecular Devices) at 37°C for 12 hours and the

absorbance of the culture determined by reading in OD

620nm every 30 minutes. The percentage of growth inhi-

bition was calculated using the GraphPad 6.01 program

(GraphPad Software) as a tool using the formula (1-SG

/ CT) x 100, with SG corresponding to the area under

the

growth curve of bacteria treated with artificial gastric

juice

and CT, the control. The tests were performed in triplicate,

with two repetitions.

Results and discussion

Regarding microorganisms’ count (Table 1) it

was

possible to observe if counts were between 10

to 10

CFU/ml.

According to Vinderola and Reinhemer (2003),

recomendend

dose would be approximately 10

CFU per

dose. Smaller

values are accepted, once their effective- ness is proven

(Minelli and Benini, 2008). Considering the approximate

consumption of 100g of product, the product with the

lowest count would be able to achieve at least the necessary

count for its functional claim.

Cad. Ciênc. Agrá., v. 13, p. 01–09, DOI: https://doi.org/10.35699/2447-6218.2021.35380

In vitro evaluation of the probiotic potential of microorganisms isolated from functional commercial fermented milks

Table 1 – Average count in Log10 (CFU/mL on MRS agar) of lactobacilli isolated from functional fermented milks

Sample

MRS count in Log

(CFU/mL)



SD *

8,780,02

8,600,01

8,400,07

7,280,02

6,340,02

5,960,04

YA – L. casei Shirota (Yakult)

AC – L. casei Defensis (Actimel)

CH – L. paracasei ST11 (Chamyto)

IT – L. casei (Itambé)

VI – L. casei (Vigor)

BA – L. acidophilus (Batavo)

* SD = Standard Deviation.

According to Forssten and Ouwehand (2020), cell

viability is the fundamental test for assessing the

effectiveness of lactic acid bacteria as probiotics. Pro-

biotic bacteria must be able to withstand stress from

gastrointestinal tract, such as gastric pH and bile salts in the

small intestine. These bacteria need to resist such injuries to

be able to exercise their therapeutic benefits.

2.0). Similar results were found by Hoque et al. (2010),

where all isolated and evaluated microorganisms were able

to survive at pH 2.2. Costa et al. (2013), in a similar study,

found a low percentage of inhibition to artificial gastric juice

and a high percentage of inhibition to bile salts, pointing the

satisfactory results of this study. Ma- ragkoudakis et al.

(2006) also observed that, within two hours, with pepsin,

artificial gastric juice did not have a great effect on the count

of probiotic microorganisms in commercial functional

fermented milks.

Regarding in vitro resistance test to artificial

gastric juice, it was possible to observe that all samples

(Table 2) were able to survive artificial gastric juice (pH

Table 2 – Percentage (%) of in vitro inhibition of gastric juice (pH 2.0) and bile salts (0.3% Oxgall) of lactobacilli iso- lated from

functional fermented milks

In vitro inhibition (functional test)

Samples

Gastric juice (%)

Bile salts (%)

No inhibition

35,77

39,12

41,36

45,31

45,71

43,77

Table 2 showed, when tested by turbidimetry in

spectrophotometry, something around inhibition of about

40% of the growth of lactobacilli with probiotic

claim

isolated from fermented milks. In the plating before

and after

exposure to bile salts at 0.3%, we can see an even more

encouraging result, with a low reduction in

counts (average

of 13% reduction – Table 3). With regard

to artificial gastric

juice (pH 2.0), there was also good

resistance to this TGI

challenge (average of 8% reduction

– Table 3). Silva et al. (2013) found higher percentages of

inhibition from bile salts of lactobacilli candidates. Costa

et al.

(2013), in turn, observed in samples of lactic acid bacteria

evaluated as to their potential, mostly, sensiti- vity to bile

salts. This reinforces the individuality of the probiotic

potential of the sample, despite the similarity

of the tested species. Caillard and Lapointe (2017) found

samples of Lactobacillus spp. with probiotic potential

tolerant and not tolerant to gastric acid (pH 2.0)

The ability of certain samples of lactic acid bac-

teria, such as Lactobacillus casei, to survive in acidic envi-

ronments is extremely important for the functionality of these

microorganisms during bioprocessing. Acid adap-

tation

experiments with L. casei ATCC 334 demonstrated

that the

induction of acid tolerance response can be triggered by

transient exposure to various sub-lethal pH values. When

faced with acid stress, bacteria can act to counter the influx

of protons, increasing the rigidity and compactness of the

cytoplasmic membrane (Broadbent et al., 2010).

Cad. Ciênc. Agrá., v. 13, p. 01–09, DOI: https://doi.org/10.35699/2447-6218.2021.35380

Campos, J. V. F. et al.

Table 3 – Percent inhibition (CFU/mL and %) of six samples of lactic acid bacteria isolated from commercial fermented milks, in

gastric pH (2.0) and bile salts (0.3% Oxgall)

Count (CFU/mL) - Percentage reduction (%)

Samples

Initial

Artificial gastric juice

Bile salt

3,95x109

6,00x109

6,55x109

5,55x109

5,30x109

5,05x109

9,10x108 - 8,72

8,87x108 - 6,64

9,13x108 - 8,04

9,13x108 - 8,49

9,70x108 - 7,58

7,80x108 - 8,36

2,82x108 - 14,48

3,25x108 - 11,94

2,48x108 - 13,21

2,86x108 - 12,96

2,30x108 - 14,01

2,24x108 - 13,94

Another factor that has an important effect on the

gastric tolerance of some samples is the presence of

milk

proteins, alone and/or in combination. Studies have shown that

L. casei 212.3 was able to survive gastric juice

in the presence

of sodium caseinate, protein whey and a combination of

these (Charteris et al., 1998b).

However, presence of resistance, if acquired (not intrinsic)

brings concern, as it may be involved in transference of

resistance of antimicrobials of clinical use, such as ce-

phalosporins, to gut pathobiont bacteria (Sharma et al.,

2014). For the other antimicrobials tested (streptomycin

and

cefoxitin), there was a diverse susceptibility profile.

Divergent results were observed by Cebeci and Gurakam

(2003), in which only 27% of the evaluated Lactobacillus

plantarum samples were resistant to gentamicin. Costa et

al.

(2013) revealed that vancomycin resistance occurred in all

samples of Lactobacillus spp. tested, which was also

observed in other studies (Anisimova; Yarrulina,

2019; Guo

et al., 2017), corroborating the statement that

lactobacilli can

have intrinsic resistance to vancomycin (Teuber et al.,

1999).

The in vitro antimicrobial susceptibility test per-

formed on some lactic acid bacteria (Table 4) showed that all

microorganisms were resistant to three antimicrobials

(ceftriaxone, gentamicin and vancomycin) and sensitive

five others (ampicillin, ciprofloxacin, chloramphenicol,

penicillin and tetracycline). It is desirable that a probiotic

candidate is sensible to all antimicrobials, in order to

avoid

genetic transference do pathogenic microorganisms.

Table 4 – Antimicrobial susceptibility profile of six samples of lactic acid bacteria isolated from commercial fermented milks

Antimicrobial

Samples

TET

AMP

EST

CRO

CIP

CFO

GEN

VAN

PEN

TET = tetracycline, AMP = ampicillin, EST = streptomycin, CRO = ceftriaxone, CLO = chloramphenicol, CIP = ciprofloxacin, CFO = cefoxitin, GEN =

gentamincin, VAN = vancomycin, PEN = penicillin. S = sensitive, M = moderately sensitive, R = resistant.

The in vitro antagonism test (from supernatant)

(Table 5) showed antagonistic activity of all samples

evaluated against the three pathogenic bacteria tested

(Escherichia coli, Salmonella Typhimurium and Staphylo-

coccus aureus). In sample BA there was a higher percentage

antagonism (77.21%) against E. coli, while sample YA

showed a high percentage of inhibition (86.65%) against

Salmonella Typhimurium. In turn, samples CH and VI

showed 72.85 and 72.66% inhibition against S. aureus,

respectively. Results by Silva et al. (2013) also showed

strong antagonistic activity against seven pathogenic

bacteria tested (which included pathogenic samples of the

species selected here), as well as those of Marag- koudakis

et al. (2006), who observed the inhibition of certain

probiotic samples of Lactobacillus spp., as well as pathogenic

E. coli and S. Typhimurium.

Cad. Ciênc. Agrá., v. 13, p. 01–09, DOI: https://doi.org/10.35699/2447-6218.2021.35380

In vitro evaluation of the probiotic potential of microorganisms isolated from functional commercial fermented milks

Table 5 – Percentage (%) of antagonism (inhibition) by the supernatant (in MRS) of lactobacilli isolated from func- tional

fermented milks against relevant pathogenic microorganisms

Inhibition of growth*

Sample

pH + SD*

E. coli

Typhimurium

S. aureus

3,960,10

4,080,06

4,000,09

4,040,07

3,960,08

4,030,11

76,88

70,72

73,35

69,50

72,08

77,21

86,65

82,74

83,72

82,27

84,15

81,72

71,92

70,16

72,85

56,85

72,66

70,82

*Inhibition values are represented as a percentage (%). SD = Standard Deviation.

Some strains of lactobacilli are capable of pro-

ducing potent antimicrobial compounds, such as bacte-

riocins, antimicrobial peptides and organic acids, such as lactic

acid (Mohanty; Saini; Mohapatra, 2017), which can

inhibit

bacteria with a deteriorating and/or pathogenic character,

including those from the following genera:

Salmonella,

Escherichia, Pseudomonas and Staphylococcus

(Castro et al.,

2011).

against S. aureus. Guedes Neto et al. (2005), in turn,

found

a diversified response on the part of the lactobacilli prospecting

regarding its probiotic characteristics. These

data reinforce

that, in the context of probiotic potential against the

inhibition of pathogens, lactobacilli tested in vitro in the

present study were able to inhibit patho- genic

microorganisms of relevance to food and diseases associated

with food, especially those of animal origin.

The in vitro antagonistic effect, by the spot on the

lawn technique, of samples of lactic acid bacteria against

pathogenic microorganisms (Escherichia coli,

Staphylococcus aureus and Salmonella Typhimurium) was

also measured, showing that all tested samples were able

produce halos of inhibition against pathogens (Graph 1).

Conclusion

Microorganisms carried by commercial fermented

milks confirmed, as expected, a favorable probiotic poten-

tial.

Tested samples presented outstanding resistance do

in vitro

gastric environment and great resistance to biliary

salts in vitro

challenge (average of 40% of inhibition). Intestinal injury

challenge did not reduce more than one

Log

of probiotic

population, which is desirable. Samples

performed

remarkable in vitro antagonism against pa- thogenic tested

strains. Resistance to cephalosporins is a

concern since they

are probably not intrinsic, with the risk of horizontal

transmission to pathobiont microorganisms.

So, probiotic

features of commercial strains performed accordingly, with

a discrete quantitative superiority of samples from Actimel,

Chamyto and Yakult.

Average of inhibition halos for each pathogenic

bacteria were more expressive over Staphylococcus aureus

and

Escherichia coli. Salmonella Typhimurium, in turn, showed

a quantitative lower inhibition by all probiotic tested

samples, which is not necessarily a sign for lower

effectiveness. Slightly different results were found by Costa

et al. (2013), in which the lowest quantitative inhibition

against pathogenic microorganisms occurred

Cad. Ciênc. Agrá., v. 13, p. 01–09, DOI: https://doi.org/10.35699/2447-6218.2021.35380

Campos, J. V. F. et al.

Graph 1 – Average results of in vitro antagonism tests (mm of inhibition halo) of probiotic lactic acid bacteria against

pathogenic microorganisms

EC = Escherichia coli; SA = Staphylococcus aureus; ST = Salmonella Typhimurium. Bars represent standard deviation. Sample 1: BA, Sample 2: YA, Sample

3: VI, Sample 4: CH, Sample 5: IT, Sample 6: AC.

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