Selection of lactic acid bacteria with probiotic potential from colonial cheeses

[Seleção de bactérias lácticas com potencial probiótico provenientes de queijos coloniais]

ABSTRACT

The aim of the present study was to isolate and identify, through phenotypic analyses, lactic acid bacteria from samples of colonial cheeses, with probiotic potential, as well as to evaluate their antimicrobial effects, their tolerance to acidic conditions and their resistance to gastrointestinal tract in a simulated form. Ten samples of colonial cheese (n=10) were used to isolate 100 bacterial cultures. The cultures were phenotypically characterized and tested for resistance to different temperatures, carbohydrate fermentation capacity and growth capacity at different NaCl concentrations. Subsequently, 20 cultures were selected for analysis of antimicrobial activity and susceptibility, tolerance, and resistance to acidic media. It was observed that most of the bacteria presented the form of Gram-positive bacilli (67%) and negative catalase (97%), all showed growth at the evaluated temperatures (10°C and 45°C) and in relation of carbohydrate fermentation, 93% fermented glucose, 99% mannitol and all fermented lactose and sorbitol with gas production, characterizing themselves as heterofermentative. Regarding resistance to different antimicrobials, 75% were resistant to two or more antimicrobials. The survival rate for simulated conditions of the gastrointestinal tract was 15% in acidic medium (pepsin and pH 2) and 90% when acidic medium was associated with milk (pepsin + milk and pH 2), after 240 minutes. They showed longer survival time and excellent resistance in intestinal conditions. It is possible to infer that the LAB present in the colonial cheeses analyzed present promising characteristics to be considered as potentially probiotics.

Keywords:
microorganisms; phenotypic characterization; microbiota; fermented foods; antagonism

RESUMO

O objetivo do presente estudo foi isolar e identificar, por meio de análises fenotípicas, bactérias ácido-lácticas de amostras de queijos coloniais, com potencial probiótico, bem como avaliar seus efeitos antimicrobianos, suas tolerâncias às condições ácidas e suas resistências ao trato gastrointestinal de forma simulada. Dez amostras de queijo colonial (n=10) foram utilizadas para isolar 100 culturas bacterianas. As culturas foram caracterizadas fenotipicamente e testadas quanto à resistência a diferentes temperaturas, à capacidade de fermentação de carboidratos e à capacidade de crescimento em diferentes concentrações de NaCl. Posteriormente, 20 culturas foram selecionadas para análise de atividade e de suscetibilidade antimicrobiana, tolerância e resistência a meios ácidos. Observou-se que a maioria das bactérias apresentou formato de bacilos Gram-positivos (67%) e catalase negativa (97%), todas apresentaram crescimento nas temperaturas avaliadas (10°C e 45°C) e em relação à fermentação de carboidratos, 93% fermentaram glicose, 99% manitol e todas fermentaram lactose e sorbitol, com produção de gases, caracterizando-se como heterofermentativas. Quanto à resistência a diferentes antimicrobianos, 75% demostraram-se resistentes a dois ou mais antimicrobianos. O índice de sobrevivência às condições simuladas do trato gastrointestinal foi de 15% em meio ácido (pepsina e pH 2) e de 90%, quando o meio ácido foi associado ao leite (pepsina + leite e pH 2), após 240 minutos. Apresentaram maior tempo de sobrevivência e ótima resistência em condições intestinais. É possível inferir que as BAL presentes nos queijos coloniais analisadas apresentam características promissoras para serem consideradas como potencialmente probióticas.

Palavras-chave:
microrganismos; caracterização fenotípica; microbiota; alimentos fermentados; antagonismo

INTRODUCTION

The colonial cheeses are commonly produced in the Southwest region of the State of Paraná. The production techniques are transmitted verbally from generation to generation. Due to the handling techniques and, since they are produced, in most cases, with raw milk and without the addition of an initial inoculum, they have a diversified unwanted microbial population (Hermanns et al., 2014).

In fermented products some bacteria are capable of inhibiting both spoilage as well as pathogenic microorganisms, thus extending shelf-life and increasing safety of these products (O’Sullivan; Cotter, 2017; Pehrson et al., 2020).

In addition to undesirable microorganisms, lactic acid bacteria (LAB) are naturally present in these cheeses, which can present themselves in the form of cocci, coccobacilli or Gram-positive bacilli, catalase-negative, non-endospore-forming, phylogenetically distinct, immobile, facultative anaerobic, capable of performing fermentation in anaerobiosis as well as in aerobiosis and which have lactic acid as the main product of carbohydrate fermentation. In homofermentative LAB, they ferment sugars to produce mainly lactic acid under anaerobic conditions. In heterofermentative LAB, sugars are fermented to produce ethanol, CO₂, and less lactic acid (Ayyash et al., 2018; Amelia et al., 2021).

LABs are essential for the fermentation process, one of the oldest forms of preservation, due to the reduction of pH and consequent production of organic acids, such as lactic acid, from the fermentation of available carbohydrates (CHO), becoming the main antagonistic effect against different microorganisms (Mora-Villalobos et al., 2020). Aiming at the probiotic potential, the LAB have been incorporated into cheeses, as they have advantages as it creates a buffer against the highly acidic environment in the gastrointestinal tract. In addition, the dense matrix and relatively high fat content of cheese may offer additional protection to probiotic bacteria in the stomach, enhancing probiotic survival throughout gastric transit (Sharp et al., 2008; Karimi et al., 2012; Silva et al., 2015).

Among the benefits generated by the probiotic effect of LAB can be highlighted the antioxidant and antimicrobial and activity (Feng and Wang, 2020; Sadeghi et al., 2022), a better use of lactose and relief of the symptoms of intolerance to this sugar (Oliveira et al., 2022), the cholesterol levels reduction (Bhat; Bajaj, 2020), the improvement in the absorption of some nutrients (Mathur et al., 2020), the intestinal infections control, the anticarcinogenic effect and the increased immune response due to the production of antibodies (Ren et al., 2021).

Considering the important role of LAB on the human organism, it highlights the need to develop studies that can characterize the bacterial colonization of colonial cheeses, particularly these bacteria, in order to encourage the consumption of regional products as well as favor the local economy.

Thus, the aim of the present study was to isolate and identify, through phenotypic analyses, lactic acid bacteria from samples of colonial cheeses, with probiotic potential, as well as to evaluate their antimicrobial effects, their tolerance to acidic conditions and their resistance to gastrointestinal tract in a simulated form.

MATERIAL AND METHODS

Samples of colonial cheese (n=10) were purchased from Francisco Beltrão supermarkets, a city located in the southwest of Paraná state. The samples were obtained under usual packaging conditions. They were placed in a thermally insulated container and sent to the Microbiology Laboratory, in the Health Sciences Center from the State University of Western Paraná - UNIOESTE, Francisco Beltrão Campus - PR. Where they remained refrigerated until the moment of microbiological analysis.

Samples (25 g) of each colonial cheese were weighed and transferred, aseptically, to vials containing 225 mL of sterile saline solution (0.85%, pH 7). After the serial decimal dilution (10^-1, 10^-2 e 10^-3), a 100 µL aliquots were plated on agar De Man, Rogosa e Sharpe (MRS, Neogen Corporation®) and incubated at 37°C for 48 h, in anaerobic. The colonies were enumerated and ten colonies (n=10) from each sample of colonial cheese that presented distinct morphotypes were collected and transferred individually to MRS broth in test tubes and incubated at 37°C for 24 hours, totaling 100 LAB isolates. All isolates were preserved in Brain Heart Infusion (BHI) + glycerol (25%) a -20°C for further characterization.

All isolates were tested for Gram stain, cell morphology and catalase reaction. Those isolates classified as Gram-negative and catalase-positive were excluded (Cogan et al. 1997; Hermanns et al., 2014).

The isolates were tested for their capability to multiply at 10°C and 45°C. In tubes containing 10 mL of MRS broth, they were inoculated with 100 µL of active culture from different LAB isolates. The tubes were incubated at 10ºC or 45ºC for 48h. As a control, the same LAB isolates were grown in MRS broth at 37ºC for 48h, at pH 7. The bacterial growth was verified after 24 and 48h. The turbidity degree between the control and test tubes was visually compared. The experiment was carried out in triplicate, according to Adikari et al. (2021) with modifications.

The fermentative profile of the LAB isolates was evaluated by the capability to ferment the carbohydrates glucose, lactose, sorbitol, and mannitol, with gas production. In tubes containing 10mL of minimal medium (10g of Kasvi® bacteriological peptone, 5g of NaCl Neon Comercial LTDA®, 0.3g of dibasic potassium phosphate (K2 HPO4) Vetec Química Fina®, 0.0018g of phenol red Vetec Química Fina®) and 5g of the carbohydrate to be tested were inoculated with 100 µL of active culture from different LAB isolates, incubated at 37°C for 48h. To observe microbial growth and gas production, inverted Durhan tubes were added into the culture tubes. The isolates in which the tubes had gas production were characterized as heterofermentative LAB, they ferment sugars to produce ethanol, CO₂, and less lactic acid (Ayyash et al., 2018; Amelia et al., 2021). The experiment was carried out in triplicate, according to Adikari et al. (2021) with modifications.

The isolates were tested for growth capacity at concentrations of 4% and 6.5% NaCl. In tubes containing 10 mL of MRS broth added with 4% or 6.5% NaCl were inoculated with 100 µL of active culture from different LAB isolates. The tubes were incubated at 37ºC for 7 days. As a control, the same LAB isolates were grown in MRS broth, at 37ºC, for 7 days, at pH 7, without addiction of NaCl. Bacterial growth was checked every 24 hours, visually comparing the degree of turbidity between the control and test tubes. The experiment was carried out in triplicate, according to Adikari et al. (2021) with modifications.

From the Gram stain tests, catalase tests, temperatures resistance, resistance to different concentrations of NaCl and fermentation of carbohydrates 20 isolates of LAB were selected to perform the other tests. As inclusion criteria, two isolates from each colonial cheese were chosen. They are cocci or bacilli, Gram-positive, catalase-negative, fermenters of all tested carbohydrates, resistant to different concentrations of NaCl and they multiplied at the different temperatures tested.

The 20 isolates from colonial cheeses were submitted to the microbiological identification test using the system Matrix Associated Laser Desorption-Ionization - Time of Flight (MALDI-TOF). The samples were sown by the depletion technique on MRS agar to obtain isolated colonies, after that, they were immediately sent via Sedex to the AQUACEN laboratory, from Veterinary School of Federal University of Minas Gerais (UFMG) for the identification of the isolates by using the MALDI -TOF methodology (Benagli et al., 2011).

The inhibitory activity of LAB was verified by the formation of an inhibition halo on the indicator microorganisms: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 (provided by Unisep - Teaching Union of the Southwest of Paraná) and Salmonella Typhimurium ATCC 14028 (provided by Oswaldo Cruz Institute). After activation of the 20 LAB isolates, in BHI broth for 24h at 37°C, 2µL of each culture was inoculated on the plate surface containing MRS agar, 2µL of each culture, at 5 different points on each plate, so that colonies were formed. The Petri dishes were incubated at 37°C for 24h.

The indicator microorganisms were activated in BHI broth at 37°C for 24 hours. Aliquots of 100 µL of the culture medium containing the indicator microorganisms were transferred to test tubes with 10mL of BHI broth. Where a serial dilution up until 10^-2 was performed and then 750µL of the final volume was pipetted and transferred to a tube with 10 mL of BHI 0.87% agar (semi-solid agar), pre-prepared and kept liquefied in a water bath at 45ºC. Its contents were transferred into the MRS agar plates, where the LAB colonies isolated from samples had been formed. After complete solidification of the BHI semi-solid agar overlay, the plates were returned to the culture oven, where they remained for another 24 to 48 hours. The presence of an inhibition halo in the culture medium (≥5 millimeter) was considered an indicator of the production of inhibitory substances produced by LAB. The experiment was carried out in duplicate.

Susceptibility to antimicrobials was assessed by the diffusion test on Müller-Hinton agar (MHA), carried out according to the standards of the Clinical and Laboratory Standards Institute (Performance…, 2005). After cultivation on MRS agar at 37ºC for 24h, the LAB colonies were suspended in a sterile saline solution (0.85%) until a turbidity compatible with the 0.5 degree of the MacFarland scale (1 x 10⁶ CFU/mL) was obtained. Each suspension was inoculated with the aid of a swab on the surface of plates containing MHA. After drying the agar surface, paper discs were aseptically added with the aid of tweezers. They were impregnated with the following antimicrobials: azithromycin (15µg), clindamycin (2µg), chloramphenicol (30µg), ampicillin (10μg), sulfazotrim (25µg), amoxicillin (10µg), erythromycin (15µg), levofloxacin (5µg), norfloxacin (10µg), amikacin (30µg). The plates with the antimicrobials were incubated in a bacteriological oven at 37ºC for 24h. The zones of inhibition diameters were measured using a caliper. The experiment was carried out in duplicate.

To analyze tolerance to acidic conditions, twenty isolates were inoculated in BHI broth at 37°C for 24h. The resistance in different acidic conditions was evaluated in MRS broth (Oxoid (Basingtoke, UK), pH 7.0, adjusted to pH 2.0, 3.0 and 4.0 with HCl, pH 7 was used as a control. Tubes containing 10 mL of acidified MRS broth were inoculated with 0.1 mL of active culture from different LAB isolates and incubated at 37ºC. After exposure to acidic conditions of 0, 2 and 4h, serial dilutions were made up to 10^-6 of each time. And 0.1mL of the 10^-6 dilution were plated on agar and the plates were incubated at 37°C for 24h. As a control, the same LAB isolates were cultivated in MRS broth, at 37ºC for 24h and pH 7. The viable cell count was performed was counted and expressed using logarithmic notation, as Colony-forming unit per mL (CFU.mL^-1).

The twenty isolates with resistance to acid were selected for evaluation of tolerance to upper gastrointestinal transit, in a simulated condition (Huang and Adams 2004). Bacterial cells with 24 h of incubation in BHI broth at 37^oC were collected by centrifugation (4.000 × g for 5 min), washed twice with phosphate buffer pH 7.0 and resuspended in 5mL of 0.5% saline solution. An aliquot of 0.2mL of cell suspension was mixed with 0.3mL saline solution and 1.0 mL of simulated gastric or intestinal juices and incubated at 37C for 48 h. The simulated gastric fluid consisted of 3 mg/mL pepsin (Sigma) and pH 2.0, while simulated intestinal juice was composed of 1mg/mL pancreatin (Sigma), pH 8.0, with and without addition of 0.5% bile salts (Sigma). The presence of food effect on the survival during transit gastric pH 2.0 was evaluated in the same manner but substituting saline by 10% (w/v) skimmed milk. The viable cell count was performed at time 0 and after 90 and 240 min to evaluate tolerance to simulated gastric fluid, and intestinal fluid, plating 100 µL of the culture in petri dishes containing MRS agar. The data were expressed as CFU.mL^-1 values. The experiment was carried out in duplicate (Hermanns et al., 2014).

The results were statistically analyzed by means of the calculation of mean, standard deviation, analysis of variance and Tukey test with significance at the level of 5% (p<0.05) using the Sisvar program (Ferreira, 2011).

RESULTS AND DISCUSSION

Of the 100 LAB isolates analyzed, 67% were bacilli, 100% were Gram-positive and 97% catalase negative, characteristic from bacteria to the genus Lactobacillus spp. Almeida Junior et al. (2015) and Lima et al. (2022) observed, when evaluating the presence of LAB in cheeses, that the isolates had morphology of bacilli and cocci, they were Gram-positive and catalase negative.

In relation to the multiplication capacity at different temperatures, all isolates developed at 10°C and 45°C after 48 hours of incubation. The ideal temperature for the growth of lactic acid bacteria varied from 10°C to 45°C, according to the LAB species (Axelsson, 2004).

As for the ability to ferment carbohydrates, it was found that 93% of the tested isolates fermented glucose, 99% mannitol and gas formation, and all fermented lactose and sorbitol with gas production, what characterized them as heterofermentative. It is important to highlight that homofermentative bacteria, from glucose fermentation, produce only lactic acid, while heterofermentative bacteria produce ethanol, CO_2, lactic acid (Ayyash et al., 2018; Amelia et al., 2021), in addition to diacetyl and acetaldehyde, that contribute to flavor and aroma of the final product (Carr et al., 2002; Jay 2005).

All isolates when exposed to different concentrations of NaCl (4% and 6.5%) were able to multiply. The turbidity and cell mass deposit were being observed at the bottom of all tubes, when compared to control tubes. Prabhurajeshwar and Chandrakanth (2017) when evaluating LAB tolerance to different NaCl concentrations observed that Lactobacilli from curd samples were able to tolerate 1 and 6% NaCl.

Almeida Júnior (2015) when selecting LAB of artisanal goat cheese and autochthonous milk found that all isolates were tolerant to the concentration of 4% and 6.5% NaCl, as showed in this study. This characteristic is fundamental in the industrial application of LAB, especially in the cheese fermentation process, since these microorganisms must tolerate and remain viable to stressful conditions, such as acidity, temperature, salinity, and freeze drying (Bremer and Kramer, 2000).

After the preliminary tests, 20 isolates were selected to continue the following tests. Using Maldi-Tof analysis, isolates 1, 5, 6, 7 and 16 were identified as Levilactobacillus brevis. Isolates 2 and 9 as Enterococcus faecium. Isolates 8, 11 and 13 as Pediococcus acidilactici, and isolate 19 as Lacticaseibacillus rhamnosus. Isolates 3, 4, 10, 12, 14, 15, 17, 18 and 20 were not identified, because when they were transported by express shipping service (Sedex), in disposable Petri dishes, from Paraná to Belo Horizonte, they grew over the entire surface of the agar without the formation of individual colonies, making identification unfeasible.

Similar to the present study, in which MALDI-TOF was used to identify the microorganisms present in the evaluated cheeses, Angeletti et al. (1998) used the same method in the quality control of buffalo mozzarella cheese. Moreover, Kanak and Yilmaz (2019) used the method in the identification and detection of the antimicrobial activity of lactic acid bacteria isolated from local cheeses. It is important to highlight that in addition to its use in the detection of microorganisms, MALDI-TOF can be applied in the analysis of lysozyme present in cheese and in the identification of the lipid profile of cheese (Schneider et al., 2010; Damário et al., 2015).

Antagonistic activity (Table 1) was demonstrated by the 20 isolates evaluated in this study, with emphasis on isolates 1, 10, 12 and 18 that inhibited the three indicator bacteria tested, including the Gram-negative bacteria Escherichia coli and Salmonella Typhimurium. It was observed that 45% of the LAB isolates showed inhibition of the pathogen Staphylococcus aureus (1, 6, 7, 8, 10, 12, 13, 14, 18), 65% inhibited Escherichia coli (1, 2, 3, 8, 9, 10, 11, 12, 15, 16, 18, 19, 20) and 55% inhibited Salmonella Typhimurium (1, 2, 4, 5, 6, 10, 12, 13, 14, 17, 18).

LAB have been used in foods as natural preservatives, due to their power to inhibit several deteriorating and pathogenic microorganisms. In this study, the LAB showed a positive effect on inhibiting of Staphylococcus aureus, Escherichia coli e Salmonella Typhimurium. Hermanns et al. (2014), when testing isolates de LAB for antagonistic activity against indicator bacteria, observed antimicrobial activity against Escherichia coli ATCC 8739, Listeria monocytogenes ATCC 7466, Staphylococcus aureus ATCC 1901 and Salmonella Typhimurium ATCC 13076.

Almeida Junior et al. (2015) also evaluated the antimicrobial activity of LAB against pathogenic microorganisms and observed that greater numbers of LAB isolates inhibited Listeria monocytogenes and the isolated UNIVASF CAP 123 showed higher antibacterial activity against Escherichia coli.Martín et al. (2022) selected and characterized lactic acid bacteria with activity against Listeria monocytogenes from traditional RTE ripened foods. It is possible to conclude that the antimicrobial substances produced are bacteriocins. Bacteriocins are found in Gram-positive and Gram-negative bacteria. However, bacteriocins produced by Gram-positive bacteria are of particular interest, due to the industrial use of several strains belonging to this group, especially lactic acid bacteria (LAB), which have the generally recognized as safe (GRAS) status (Muñoz et al., 2011).

Table 1
Analysis of the antimicrobial activity of LAB isolated from colonial cheese, expressing the Mean ± Standard Deviation of the inhibition halos in millimeters

Isolated	Staphylococcus aureus	Escherichia coli	Salmonella Typhimurium
1	13.6±1.15	13.1±3.32	11.5±3.40
2	-	26.2± 3.54	13.8±1.93
3	-	12.5±1.67	-
4	-	-	19.5±3.16
5	-	-	15.8±1.11
6	13.7± 3.54	-	14.9±2.95
7	12.1±1.90	-	-
8	16. ±7.07	19.3±3.29	-
9	-	1.74±2.05	-
10	13.9±1.01	15.9±3.46	11.1±0.86
11	-	13.5±2.75	-
12	15.4±3.26	13.2±3.35	13.4±4.27
13	13.6±1.15	-	10.6±1.00
14	16.2±3.70	-	27.0±5.05
15	-	12.5±3.34	-
16	-	12.6±2.35	-
17	-	-	17.6±4.02
18	11.7±1.88	17.1±4.01	10.8±1.73
19	-	13.1±3.32	-
20	-	15.7±5.23	-

As well as the production of organic acids, hydrogen peroxide and substances with bactericidal or bacteriostatic actions can also happen during lactic fermentation. Thus, they can exert antagonistic activity against the growth of pathogenic and deteriorating bacteria in food (Magnusson and Schnurer, 2001; Reis et al., 2012; Halimi and Mirsalehian, 2016; Muhammad et al., 2019; Tarrah et al., 2019; Chen et al., 2022). For this reason, there is a great interest in the use of LAB in foods with ingredients that favor the human and animal microbiota (Oliveira et al., 2022; Sadiq, 2022; Khushboo et al., 2023).

A good probiotic should not carry antimicrobial resistance genes (Montoro et al., 2018), i.e., although many LAB strains, especially those of Lactobacillus spp., are resistant to certain antimicrobials, this resistance is usually non-transferable (Rotta et al., 2020). The antimicrobial susceptibility test (Table 2) demonstrated that the strains of Lactobacillus spp. were resistant to azithromycin (55%), clindamycin (85%), chloramphenicol (15%), ampicillin (40%), sulfazotrin (95%), amoxicillin (25%), erythromycin (20%), levofloxacin (15%), norfloxacin (10%), amikacin (30%). In contrast, some isolates were sensitive to the antimicrobial azithromycin (40%), chloramphenicol (85%), ampicillin (40%), sulfazotrin (5%), amoxicillin (75%), erythromycin (80%), levofloxacin (15 %), norfloxacin (10%), amikacin (30%), and none of the isolates showed sensitivity to clindamycin. Furthermore, the strains showed intermediate resistance to azithromycin (5%), clindamycin (15%), norfloxacin (10%), amikacin (10%).

Regarding the different classes of antimicrobials tested, all had 2 or more resistant isolates. The beta-lactams class and sulfonamides were the ones that showed the greatest resistance of the isolates. Both classes obtained a total of 95% of the isolates resistant to at least one of the class antimicrobials. The lincosamides showed a total of 85% of resistant isolates, and the macrolides class added 75% of isolates resistant to their antimicrobials. In contrast, the quinolones class showed only 25% of resistant isolates, followed by the class of amphenicols, which showed the least resistance on the part of isolates, only 15% resistant.

Table 2
Result regarding the resistance of LAB strains isolated in the study to different types of antimicrobials

Antimicrobials tested
Isolated	Azi	Cli	Clo	Amp	Sut	Amo	Eri	Levo	Norf	Ami
	(15µg)	(2µg)	(30µg)	(10µg)	(25µg)	(10µg)	(15µg)	(5µg)	(10µg)	(30µg)
1	R	R	S	S	R	S	S	R	S	R
2	I	R	S	R	R	R	R	S	S	R
3	R	R	S	R	R	R	R	S	S	S
4	S	R	S	R	R	S	S	S	S	S
5	S	R	S	S	R	S	S	S	S	S
6	R	R	R	R	R	R	R	S	S	S
7	S	R	S	S	R	S	S	S	R	S
8	R	R	S	S	R	S	S	S	S	R
9	S	R	S	R	R	S	S	S	I	R
10	R	R	S	R	R	S	S	R	R	R
11	S	R	S	S	R	S	S	S	S	S
12	R	R	R	R	R	R	S	S	S	S
13	R	R	R	S	R	S	S	S	S	I
14	S	R	S	R	R	R	R	S	S	S
15	S	I	S	S	S	S	S	S	S	I
16	R	R	S	S	R	S	S	R	S	S
17	R	R	S	S	R	S	S	S	S	S
18	S	I	S	S	R	S	S	S	S	S
19	R	I	S	S	R	S	S	S	I	S
20	R	R	S	S	R	S	S	S	S	R
R (%)	55	85	15	40	95	25	20	15	10	30

• Note: AZI: Azithromycin; CLI: Clindamycin; CLO: Chloramphenicol; AMP: Ampicillin; SUT: Sulfazothrin; AMO: Amoxicilin; ERI: Erythromycin; LEVO: Levofloxacin; NORF: Norfloxacin; AMI: Amikacin.
• R: Resistant; S: Sensitive; I: Intermediate.

Of the total isolates tested, 75% showed a multidrug resistance profile, showing resistance to three or more different classes of antimicrobials. On the other hand, isolate 15 was not resistant to any of the tested antibiotics, isolate 18 was resistant only to the sulfonamide class, isolate 19 was resistant to sulfonamides and macrolides, and isolates 5 and 11 were resistant only to the sulfonamide class and lincosamides.

Antimicrobial resistance is an increasingly frequent problem with worldwide spread, compromising the clinical treatment of various pathologies that affect humans and animals (Salam et al., 2023). Thus, LAB have been evaluated in order to be used as probiotics in the reconstitution of the intestinal microbiota (Oliveira et al, 2022; Latif et al., 2023), as well as a constant demand from the food industry for LAB that produce substances with antimicrobial potential, such as antimicrobial peptides, which inhibit various pathogenic and deteriorating microorganisms (Castellano et al., 2017; Latif et al., 2023).

Antimicrobial resistance is closely related to food safety and should always be investigated when there is an intention to use new strains of microorganisms in food products. Since these new strains can carry resistance genes that can be transferred to other bacteria, they increase the potential for virulence and present resistance to different antimicrobials, which can endanger human health (Probiotics…, 2006).

As in the present study, some LAB showed resistance or sensitivity to antimicrobials. Funck et al. (2019) when evaluating the action of antimicrobials on the isolate Lactobacillus curvatus P99 observed phenotypic resistance to ciprofloxacin, trimethoprim‐sulfamethoxazole, amikacin, ampicillin, and gentamicin; and sensitivity to penicillin, clindamycin, chloramphenicol, tetracycline, erythromycin, and cephalothin.

Probiotic bacteria must resist adverse conditions in the gastrointestinal tract reaching the gut in their viable form. Thus, in vitro resistance to acidic pH and bile salts are essential characteristics for a strain to be considered potentially probiotic, as they reveal a greater chance of the strain surviving passage through the stomach and duodenum in vivo (Wendling and Weschenfelder, 2013).

The analysis of the tolerance of the LAB to acidic conditions (Table 3) showed that the isolates 1, 3, 4, 5, 6, 7, 8, 9 and 10 showed survival for media with pH 2, 3 and 4. However, there was a significant reduction in the number of viable cells after 4 hours of exposure to pH 2; on the other hand, when subjected to 4 hours of exposure to pH 4, the isolates had a higher survival rate. LAB isolates 2, 12, 13, 14, 16, 17, 18, 19 and 20 survived a at 0, 2 and 4 hours when exposed to pH 3 and 4. The isolates 2 and 15 did not show viable cells after 4 hours of exposure to pH 3.

In addition to the characteristics already discussed, the capability to survive in the environment in which it will act is an essential characteristic when choosing a probiotic microorganism. For LAB strains to have a beneficial effect on intestine health, they need to be able to survive low acidic conditions (Liu et al., 2013). In the study by Solieri et al. (2014), the critical limit of survival under exposure to acidic conditions was pH 2.0, which was effective in completely inhibiting the survival of almost all the strains.

To survive in the intestine, microorganisms must tolerate the action of digestive enzymes and the low pH of the stomach, ranging from 2.5 to 3.5, reaching pH 1.5 during fasting or 4.5 when the individual is fed, and digestive enzymes. This high degree of acidity can lead to the destruction of several microorganisms ingested, since most of them are sensitive to pH values below 3. However, it is important to highlight that the nature of the food can alter the transit time in the gastrointestinal tract, which usually takes from 2h to 4h, enabling the microorganism to remain, due to its buffering and protective effect (Huang; Adams, 2004; Huang et al., 2014).

When evaluating the tolerance of simulated gastric juice with pepsin at pH 2, all isolates significantly decreased the number of viable cells after 90 minutes, and only isolates 3, 4 and 15 expressed a very low number of viable cells after 240 minutes. At the same time, when the tolerance of the isolates to the same conditions was evaluated, with the addition of a food, in the case of skimmed milk reconstituted at 10%, it was observed that 90% showed a significant growth after 240 minutes (Table 4).

The isolates were also evaluated for tolerance to artificial intestinal juices pancreatin 1mg/mL + pH 8 and pancreatin 1mg/mL + 0.5% bile + pH 8. When the isolates grew in the presence of pH 8 and pancreatin, 30% showed significant growth (2, 8, 11, 14, 16 and 19) after 240 minutes. On the other hand, only 20% grew significantly (3, 14, 19, and 20) in the presence of pancreatin, bovine bile and pH 8.

Thus, the present study found that acidic conditions are capable of interfering in the LAB activity, while the presence of food (in this case, the milk) was able to maintain its viability. Meira et al. (2010), Ranadheera et al. (2014), also observed similar results. Huang and Adams (2004) claim that the low tolerance of some strains when subjected to simulated gastric juice is not sufficient to remove their probiotic effect, since the strains can reach the intestine in high concentrations when buffered by food or encapsulated, thus promoting positive effects on human health.

It is worth mentioning that, for the product to be commercialized in Brazil with the probiotic property, the microorganisms used must have tolerance against the barriers of the gastrointestinal tract, as well as they must have a viable cells number sufficient to perform the beneficial functions to the human organism (Brasil, 2008).

Table 3
Final counts of viable microorganisms of the 20 strains submitted to cultivation at different pH values for 48 hours

Isolated	Tested times	Final countdown (N x 109 CFU.mL)
		pH 7	pH 2	pH 3	pH 4
1	0h	2.7a	1.9b	2.5c	2.0b
	2h	2.7a	0.2a	1.8a	1.2a
	4h	3.1b	0.1a	2.2b	3.0c
2	0h	3.9a	1.2b	3.2c	2.9a
	2h	3.8a	0.1a	2.2a	3.5c
	4h	4.2b	0.0a	2.6b	3.2b
3	0h	3.2b	2.3c	2.2a	3.2c
	2h	3.0a	0.3a	3.4b	2.8b
	4h	3.6c	0.7b	3.8c	1.8a
4	0h	2.9b	3.3c	3.2c	3.6b
	2h	2.6a	1.0b	2.3a	4.3c
	4h	3.8c	0.3a	2.6b	3.0a
5	0h	1.4a	3.4c	1.9a	3.0a
	2h	3.2b	0.6b	3.2c	4.0c
	4h	3.8c	0.3a	2.7b	3.5b
6	0h	3.3a	1.6c	3.0c	3.8b
	2h	3.3a	0.4b	2.1b	4.4c
	4h	3.8b	0.2a	1.7a	3.3a
7	0h	2.6a	1.7b	4.2c	3.8c
	2h	3.9b	0.7a	3.1b	2.1b
	4h	4.4c	0.7a	2.0a	1.9a
8	0h	2.6a	1.4b	2.4c	1.9a
	2h	4.8b	0.2a	1.8a	1.9a
	4h	5.0c	0.2a	2.0b	2.1b
9	0h	3.2a	1.9c	2.2a	5.0c
	2h	4.2b	0.5b	3.1b	2.2a
	4h	5.0c	0.1a	2.1a	3.1b
10	0h	3.2a	2.9c	0.9a	2.4a
	2h	4.1b	0.4b	1.8b	3.0b
	4h	4.2b	0.1a	1.8b	3.8c
11	0h	0.2a	0.3b	0.3b	0.4a
	2h	0.2a	0.0a	0.2b	0.3a
	4h	0.3a	0.0a	0.0a	0.3a
12	0h	0.4b	0.3b	0.1a	0.8c
	2h	1.2c	0.0a	0.6b	0.3b
	4h	0.2a	0.0a	0.6b	0.1a
13	0h	0.2a	0.2b	0.6b	0.7b
	2h	0.4b	0.0a	0.2a	0.2a
	4h	1.0c	0.0a	0.2a	0.4a
14	0h	0.2a	0.2b	0.4a	0.6b
	2h	0.4b	0.0a	0.4a	0.4a
	4h	0.6c	0.0a	0.3a	0.3a
15	0h	0.5a	0.1a	0.4b	2.1c
	2h	0.5a	0.1a	0.1a	0.5b
	4h	0.6a	0.0a	0.0a	0.3a
16	0h	0.4a	0.2b	0.5a	0.3a
	2h	1.6b	0.1ab	1.1b	0.4a
	4h	2.5c	0.0a	0.5a	1.6b
17	0h	0.2a	0.2b	0.5a	0.5b
	2h	2.3c	0.1ab	0.7a	0.3a
	4h	2.1b	0.0a	1.5c	1.1c
18	0h	2.1c	0.2b	0.4b	1.5c
	2h	1.2b	0.0a	0.1a	0.6b
	4h	0.4a	0.0a	0.5b	0.1a
19	0h	1.1b	0.2a	0.2a	0.1a
	2h	2.2c	0.0a	0.5b	0.3a
	4h	0.3a	0.0a	0.2a	0.1b
20	0h	0.2a	0.1a	0.3a	0.2a
	2h	0.6a	0.0a	0.2a	0.9a
	4h	0.6a	0.0a	0.8b	0.8b

• Note: CFU = Colony-Forming Unit; N: Values obtained. Mean values followed by their standard deviation. Different letters in the same column indicate a significant difference between the means by Tukey's test (p < 0.05). (CV%) = Coefficient of variation.

Table 4
Final counts of viable microorganisms from the 20 strains subjected to cultivation for 48 hours under different conditions to simulate in vitro resistance to conditions similar to the gastrointestinal tract

Isolated	Final countdown (N x 103 CFU.mL)
	Pure pepsin pH 2			Pepsin + milk pH 2			Pure pancreatin pH 8			Pancreatin + Bile bovine pH 8
	0min	90min	240min	0min	90min	240min	0min	90min	240min	0min	90min	240min
1	3.9b	0.0a	0.0a	2.3a	3.3c	2.7b	2.5b	1.5a	1.3a	2.4a	3.5b	2.8a
2	3.2c	1.1b	0.0a	2.7b	1.9a	1.5a	1.9a	3.8c	3.0b	3.8a	3.2a	3.6a
3	2.9b	0.1a	0.1a	2.8b	1.8a	1.9a	1.6b	0.9a	0.6a	3.0a	3.8b	4.0b
4	2.4b	0.1a	0.1a	1.3a	2.7c	1.9b	1.2a	0.8ab	0.5a	2.4a	3.5b	2.8a
5	2.4a	0.2a	0.0a	1.0a	1.7b	1.9b	1.4a	1.3a	1.0a	2.9b	1.7a	3.4b
6	1.3b	0.1a	0.0a	0.8a	1.4b	1.4b	2.1ab	1.8a	2.7b	1.7a	1.4a	1.5a
7	2.3b	0.1a	0.0a	0.8a	0.9a	1.0a	1.3a	2.4b	1.6a	1.3a	0.7a	0.7a
8	2.2b	0.1a	0.0a	0.9a	1.2a	1.3a	2.6a	2.1a	3.1b	0.9a	0.7a	0.8a
9	3.3b	0.1a	0.0a	1.0a	1.7b	2.1c	0.9a	0.8a	1.1a	1.9b	1.2a	1.4b
10	2.9b	0.1a	0.0a	0.9a	1.6b	2.6c	1.3a	1.7a	1.1a	1.5a	1.4a	1.3a
11	0.3a	0.2a	0.0a	0.9a	1.2a	1.3a	0.9a	1.0a	2.4b	1.0a	1.2a	0.9a
12	0.7b	0.1a	0.0a	0.7a	0.9ab	1.3b	0.7a	0.7a	0.9a	0.6a	0.9a	1.0a
13	0.3a	0.0a	0.0a	0.4a	1.0b	1.3b	0.4a	0.6a	0.9a	0.6a	0.7a	0.1a
14	0.8b	0.3a	0.0a	0.9a	1.0a	1.0a	1.3a	1.8ab	2.0b	1.0a	1.7b	1.9b
15	0.2a	0.1a	0.1a	0.2a	0.7a	1.1c	0.2a	0.5a	0.7a	0.3a	0.3a	0.6a
16	0.5b	0.0a	0.0a	0.3a	0.2a	0.9b	0.1a	0.4ab	0.9b	0.1a	0.3a	0.6a
17	0.3a	0.0a	0.0a	0.4a	0.7ab	1.1b	0.4a	0.5a	0.9a	0.2a	0.1a	0.4a
18	0.4a	0.1a	0.0a	0.2a	0.6b	1.3c	0.5a	0.7a	0.8a	0.2a	0.3a	0.6a
19	0.3a	0.1a	0.0a	0.8a	1.2b	1.5b	0.9a	1.3ab	1.9b	0.9a	1.2a	1.9b
20	0.3a	0.1a	0.0a	0.4a	1.1b	1.6c	0.4a	0.8a	1.0a	0.2a	1.1b	1.2b

• Note:CFU = Colony-Forming Unit; N: Values obtained. Mean values followed by their standard deviation. Different letters in the same column indicate a significant difference between the means by Tukey's test (p < 0.05). (CV%) = Coefficient of variation.

CONCLUSIONS

In this study, lactic acid bacteria isolated from colonial cheese have probiotic properties, including tolerance to acids and bile salts, in addition to exhibiting antimicrobial activity against important pathogenic bacteria. Isolates 1, 10, 12 and 18 showed antimicrobial activity against the three bacterial pathogens tested. Isolate 15 was not resistant to any of the antimicrobials evaluated, isolate 18 was resistant to only one antimicrobial and isolates 5, 11 and 19 were resistant to two antimicrobials evaluated.

Of the total isolates, 45% survived in culture medium with pH 2, 3 and 4 and 70% grew significantly in the presence of Pepsin + milk pH 2, however, when evaluating growth in the presence of pancreatin, bovine bile and pH 8 only isolates 3, 14, 19 and 20 grew. The results indicate that 60% of the isolates, especially isolates 1, 10 and 19, have promising characteristics to be considered as potentially probiotics. Therefore, further in vitro and in vivo studies should be conducted to identify candidate microorganisms for use as probiotics isolated from colonial cheese.

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Publication Dates

• Publication in this collection
  21 Feb 2025
• Date of issue
  Mar-Apr 2025

History

• Received
  17 Feb 2024
• Accepted
  25 Aug 2024