Phenotypic and molecular characterization of the L. lactis R7 isolate
The isolate L. lactis R7 showed typical characteristics of the LAB group: Gram-positive, catalase-negative and homofermentative. Therefore, it produced lactic acid as the final product of carbohydrate fermentation, avoiding flavor and undesirable characteristics in the final product. Many species of the Lactococcus has been widely used in the food industry, since present microbiological safety. Mostly, showed probiotic characteristics, contributing sensorially to products; they also increase the nutritive value of products through the synthesis of vitamins, proteins and essential amino acids (Giraffa 2004). As well, it is important the use of LAB in the control of pathogens through the production of metabolites with antimicrobial properties, as well as in food preservation (Ayala et al. 2019).
For the identification of isolate, it was performed phenotypic characterization (Table 1). The results obtained demonstrated that L. lactis R7 had growth potential in a wide temperature range (between 15 °C and 45 °C). The bacterial capability to grow at high temperature is a good characteristic, as it could be interpreted as indicating an increased rate of growth and lactic acid production. Moreover, a high fermentation temperature decreases contamination by other microorganisms (Ibourahema et al. 2008; Menconi et al. 2014). L. lactis R7 demonstrated also ability to tolerate high osmotic concentrations of sodium chloride at 4.0 %, and this is a characteristics of the L. lactis subsp. lactis. This result was in accordance with others studies showing that sodium chloride tolerance might be strain-dependent (Bevilacqua et al. 2010). The knowledge about sodium chloride tolerance of an L. lactis isolate allows a rational selection and control of starter cultures in the manufacture of dairy products, especially cheese, with different levels of salt. Thus, incorporation of strains more sensitive to salt in initial cultures would potentially increase autolysis and the release of intracellular enzymes, neutralizing with sensory defects, such as bitterness, often described in low-salt cheeses (Kristensen et al. 2020).
The isolate showed ability to growth in acidic and alkaline condition (pH 2.0 and 9.6). One of the most important criteria in selecting bacterial with probiotic potential is that they are tolerant to acidic conditions. For ensure that the viability and functionality to be satisfactorily exercised, probiotic bacteria need to survive at pH 1.5 to 2.0, this condition resembles the passage in the host's human stomach (Dunne et al. 2001). This characteristic will ensure survival in this habitat, together with the other probiotic characteristics described below. In the same way, L. lactis R7 showed ability to sugar fermentation: glucose, maltose and ribose. In accordance to other authors (Desmasures et al. 1998; Fernández et al. 2011) these results are important parameters because L. lactis subsp. lactis is distinct from L. lactis subsp. cremoris according to five phenotypic criteria: the ability to grow at 40 °C, in 4.0 % of NaCl, pH 9.2, the ability to ferment maltose and the ability to deaminate arginine, for all of which L. lactis subsp. cremoris strains are reported as negative. Furthermore, Drici et al. (2010) reported also that the ablitlity to resist at pH 9.6, at 45 °C of temperature, but not in the presence of 6.0% NaCl, differentiates between the Enterococcus and Lactococcus gender, because Lactococcus does not tolerate a concentration of NaCl higher than 4%. Thus, the findings of the present study confirm that the isolated L. lactis R7 belong to the genus Lactococcus.
The currently, L. lactis subspecies defined are lactis; cremoris; hordniae and structae. The strains of the subspecies lactis and cremoris are central components of the culture blends used in the commercial production of cheese, which, through lactic acid, influence the flavor, texture and quality of the final products. However, the heat sensitivity of cremoris subspecies compared with lactis subspecies often precludes their use in certain applications. The taxonomic classification of L. lactis subsp. lactis and subsp. cremoris is based on phenotype and differentiated on the basis of growth temperature, salt tolerance and arginine utilization. However, with progress in molecular methods in the last decade, it has become clear that comparison of strains from a broad range of different environmental niches challenge these phenotypic distinctions and that a combination of genotype and phenotype is required to describe strains of this species (Pérez et al. 2011; McAuliffe 2018).
For this reason, the isolate L. lactis R7 was analyzed by 16S rRNA gene. The results from molecularly identification showed that L. lactis R7 as belonging to the genus Lactococcus, species lactis, subspecies lactis (L. lactis subsp. lactis), which was deposited in GenBank under accession number KF879126, showing similarity of 99.8% with other sequences, based on the 16S rRNA gene, previously deposited in GenBank (Figure 1). The phenotypic characteristics showed in the present study combined with the molecular identification, indicate that the isolate R7 belongs to the lactis subspecies.
Probiotic characterization of the L. lactis R7 isolate
The ability to tolerate acidity has been shown by some authors to be strain-dependent (Manini et al. 2016; Flach et al. 2018; Cruxen et al. 2019). The isolate was evaluated for its ability to survive at different pH values (2.0, 3.0 and 4.0), as shown in Figure 2(A). As can be seen, L. lactis R7 showed cell viability of 7.08 log CFU.mL-1, after 4 h of cultivation at pH 2.0. At pH 3.0 and 4.0, the log reduction was 1.31 and 1.20 log CFU.mL-1, respectively, at the end of 4 h of exposure, indicating acidity tolerance. Guo et al. (2009) reported that the variation in acidity tolerance may be related to the difference in H+-ATPase activity, which controls the intracellular concentration of H+, maintaining pH homeostasis and cell viability (Meira et al. 2012).
Bile, even in low concentrations, can inhibit the growth of microorganisms, and survival in the concentration of 0.3% has been considered a critical value for the screening of isolates resistant to this compound (Manini et al. 2016). Lactococcus lactis R7 was able to maintain cell viability (7.15 log CFU.mL-1) after 4 h of exposure to bile salts at a concentration of 0.5%, as seen in Figure 2(B).
The viability of L. lactis R7 to the simulated gastrointestinal tract was evaluated in the presence and absence of food (milk). The results are shown in Figure 3(A). The cellular concentration of L. lactis R7 was reduced in the presence of pepsin (pH 2.0) over time, with a logarithmic reduction of 2.73 cycles (p < 0.05). On the other hand, when the isolate was exposed to simulated gastric fluid and the food matrix, the cell reduction was significantly lower (1.64 log CFU.mL-1), demonstrating that the food provided protection to the microorganism (p < 0.05).
Studies report that milk proteins have technological properties, such as buffering capacity, good emulsification and ability to form networks even at low concentrations, ensuring good survival of microorganisms during digestion (Ranadheera et al. 2012; Prasanna et al. 2018). Similar results were found by Hwanhlem et al. (2010), who reported that Lactobacillus strains were able to maintain their viability when exposed to acidity conditions with pH between 2.5–4.0; however, at lower pH values there was a reduction in their viability. Pieniz et al. (2014) evaluated the E. durans LAB18s isolate from fresh Minas cheese, which showed high survival capacity in the presence of simulated gastric fluids containing pepsin (pH 3.0) and simulated intestinal fluids containing pancreatin (pH 8, with or without addition of bile salts). The viability of E. durans LAB18s was also satisfactory when exposed to pH 3.0 and 4.0, although a decrease in viable cell counts was observed when pH 2 was evaluated, a result that is similar to that found in the present study.
In the simulated intestinal tract (Figure 3(B)), cell concentrations above 7.98 log CFU.mL-1 were obtained, both in the presence and in the absence of bile salts (0.5%), at all times. The exception was the time of 4 h in the presence of bile salts, where there was a significant reduction of 2.86 logarithmic cycles, reaching the minimum value so that the isolate can have beneficial effects as a probiotic (6 log CFU.mL-1). As observed in the results of bile salt tolerance (Figure 3(B)) and in the literature reports, L. lactis R7 showed activity of the bile salt hydrolase (BSH) enzyme in the small intestine. This is because the resistance to bile salts of some isolates is related to the activity of the enzyme that helps hydrolyze conjugated bile, reducing its toxicity (Ilha et al. 2015; Chen et al. 2017). These results show that this isolate has resistance to bile salts and suggest the possibility of its survival in the human gastrointestinal tract.
The probiotic potential of LAB isolated from milk and sheep cheeses was evaluated by Meira et al. (2012), who found that these bacteria were able to tolerate low concentrations of bile salts (0.1 and 0.3%). Only the LCN 56 strain (L. plantarum) presented cell concentrations above the limit of detection when exposed to 0.5% of bile salts. In addition, none of the evaluated strains presented viability above the limit of detection after the 4 hours of incubation. Considering that the concentration of bile salts of 0.15 to 0.3% has been recommended for the in vitro evaluation of the passage through the small intestine (Huang and Adams 2004), the isolate L. lactis R7 showed probiotic potential under the studied conditions.
The survival of ingested probiotics faces stomach acidity and the presence of bile salts in the duodenum as the main obstacle (Chu-Ky et al. 2014), this resistance to acids also remains in acidic food applications. However, brief and mild exposure to acids can result in greater acid resistance in future exposures (Mills et al. 2011; den Besten et al. 2013).
In addition, to the probiotic potential, L. lactis R7 also showed anticarcinogenic potential in colorectal cancer induced in animal models, in a previous study of this research group (Jaskulski et al. 2020). There are no reports so far in the literature about such effects presented by this isolate. These results are promising because, in addition to demonstrating the potential for colorectal cancer stabilization, they challenge science to explore the effects of L. lactis R7 and continue research.
Auto-aggregation, co-aggregation and hydrophobicity
The auto-aggregation capacity of the L. lactis R7 isolate increased exponentially over the evaluated time, achieving a maximum value at 4 h (25.8 ± 0.03%) of incubation and then decreasing. The same profile was verified in relation to the coaggregation capacity (18.3 ± 0.02%) of the isolate. It is known that auto-aggregation and co-aggregation among bacteria play an important role in preventing the colonization of surfaces of the intestinal mucosa by pathogens (García-Cayuela et al., 2014). It is also well known that co-aggregation abilities of LAB isolates might interfere with the ability of the pathogenic microorganisms to infect the host, being able to prevent colonization by foodborne pathogens (García-Cayuela et al. 2014).
In addition, the isolate showed 11.1% ± 0.01 of adhesion capacity in the hydrophobicity test. This is relevant data for a probiotic candidate microorganism: it is a prerequisite for adhesion to the intestinal epithelium, so that it can colonize the intestine and exert a beneficial effect on the host, by exclusion and competition for sites of link with enteropathogenic bacteria (Dlamini et al. 2019).
Phenotypic characteristics such as adhesion, self-aggregation, co-aggregation and surface hydrophobicity favor microbiota colonization. However, this is a complex mechanism, which involves many factors, but mainly aggregation and hydrophobicity allow interactions to occur between the microorganism and the host, so that it promotes beneficial health effects (García-Cayuela et al. 2014).
The antioxidant capacity of LAB is important for protection against free radicals (Ren et al. 2014). The evaluation by the TBARS method showed that the isolate presented antioxidant potential and the ability to inhibit lipid peroxidation (Figure 4A). In the DPPH method, antioxidant activity was also observed: the isolate showed the ability to sequester the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, verified by a color change from violet to yellow when it comes into contact with the antioxidant substance (Figure 4B).
Similar results were found by Pieniz et al. (2014), verified that the isolates Enterococcus sp., E. faecalis, E. faecium and E. hirae presented antioxidant capacity in both methods evaluated, TBARS and DPPH. According to Wang et al. (2012), LAB that have the property of sequestering free radicals are of great interest to the food industry, as they are able to prevent deterioration and increase the shelf life of products while maintaining their nutritional value. It should be noted that the antioxidant capacity of probiotic bacteria is strain-specific, since it is related to cell wall composition, presence of enzymes and production of different metabolites (Su Oh et al. 2018).
Production of hemolysin and activity of gelatinase, DNAse and lipase
Lactococcus lactis R7 showed satisfactory results in terms of safety: there was absence of DNAse, gelatinase and lipase activity, and hemolysin was not produced. Therefore, it does not present risks to human health (Domingos-Lopes et al. 2017).
Analysis of the results obtained for the antimicrobial susceptibility test showed that the L. lactis R7 isolate showed susceptibility for all the antimicrobials tested, according to CLSI standards (Table 2). A similar study by Pieniz et al. (2015) demonstrated that the E. durans LAB18s isolate from fresh Minas cheese presented high susceptibility for all the antimicrobials tested, thus demonstrating their safe use in food.
According to Kondrotiene et al. (2020), LAB should be investigated for their resistance to antimicrobials before being applied in commercial products, since the ability to transfer resistance genes to these compounds is a risk factor for use in food. The isolates of L. lactis showed great variation in resistance to antimicrobials, according to the cutoff point provided by the European Food Safety Authority (EFSA), for streptomycin and tetracycline.
Lactococcus lactis R7 was evaluated for its ability to form biofilm, being classified as a non-biofilm producer. Gomes et al. (2008) evaluated the prevalence of genera of LAB isolated from food. Among them, isolates from samples of ricotta cheese were evaluated, and these presented poor capacity of biofilm formation, a result similar to that found in the present study. The formation of biofilm in the gastrointestinal tract or in the vaginal mucosa can contribute to the fixation and colonization of bacteria. However, the presence of LAB biofilm in the oral cavity contributes to the appearance of cavities. Also, in the food industry, its adhesion to surfaces and foods can lead to corrosion or deterioration (Arena et al. 2017).
Antagonist activity of L. lactis R7 against selected pathogenic bacteria
The isolate L. lactis R7 showed antimicrobial activity against the pathogens tested through agar disk-diffusion test, as shown in Table 3. Four foodborne pathogens were tested and the isolate showed antagonistic activity against three. The largest halos were observed for S. aureus ATCC 25923 (12.02 mm ± 0.06) and E. coli ATCC 8739 (11.1 mm ± 0.15), while the lowest inhibition halo was found for S. enteritidis ATCC 13076 (9.5 mm ± 0.03). There was no activity against L. monocytogenes ATCC 19114.
There is growing research interest in new antimicrobial substances and/or microorganisms with antimicrobial potential. They are safe and natural alternatives obtained from LAB; also, they are promising biological control alternatives for the prevention or treatment of diseases of the digestive system (Jabbari et al., 2017). Jang et al. (2019) evaluated the antimicrobial activity of L. brevis KU15153, which demonstrated inhibitory activity against E. coli, S. aureus, L. monocytogenes and S. typhimurium. Among the pathogens, S. aureus showed a greater zone of inhibition (18 mm ± 0.3), corroborating the results of the present study. The antimicrobial activity expressed by LAB may be due to the production of metabolites, such as acid (lactic, acetic, among others), hydrogen peroxide, diacetyl, bacteriocin, among other molecules (Oliveira et al. 2017).
MTT cytotoxicity assay
The cytotoxic effects of the cell suspension were evaluated in VERO cell culture by performing the MTT assay, as shown in Figure 5. The results obtained in the treated cells showed no statistical difference (p = 0.907) in relation to the control and, even after 24 h of incubation, the metabolites resulting from the cell suspension of L. lactis R7 had not caused damage to the cells, which maintained the integrity of their membrane. Haghshenas et al. (2014) evaluated the cytotoxicity of L. lactis subsp. lactis 44 using the MTT test in HUVEC cells (cells of the human umbilical endothelium). They found that the metabolites, in the concentration of 30%, did not present cytotoxicity, corroborating the results of the present study.
The MTT assay is considered very sensitive for the determination of cell respiration, viability and cytotoxicity, as only viable cells are able to produce formazan products during the test, in addition to sustained bacterial fixation, even after several washes. The optical density of the remaining cells is able to remain high after absorbing the MTT stain (Vaucher et al. 2010; Poormontaseri et al. 2017; Yasmin et al. 2020).