In vitro evaluation of probiotic and technological properties of lactic acid bacteria isolated from artisanal cheese produced in the Serra Geral - Minas Gerais - Brazil

There is great technological potential for the use of lactic acid bacteria (LAB) isolated from artisanal dairy products, such as cheese, and it has generated an increasing interest in the study and identification of these microorganisms, in addition to the evaluation of their potential probiotic properties. Andrade et al. (2014) and Silva et al. (2019) described in vitro probiotic potential of LAB isolated from Minas artisanal cheese produced in the Canastra and Araxá regions, respectively. However, to date, there is no research in the literature on the in vitro probiotic potential of LAB isolated from artisanal cheeses produced in the Serra Geral region, as well as their raw materials and production environment. Thus, the present study aimed to evaluate the in vitro probiotic potential of LAB isolated from those cheeses, raw milk, endogenous starter cultures and swabs from surfaces. The LAB used (Table 1) were previously isolated and identified by Caldeira (2021). 

There is great technological potential for the use of lactic acid bacteria (LAB) isolated from artisanal dairy products, such as cheese, and it has generated an increasing interest in the study and identification of these microorganisms, in addition to the evaluation of their potential probiotic properties. Andrade et al. (2014) and Silva et al. (2019) described in vitro probiotic potential of LAB isolated from Minas artisanal cheese produced in the Canastra and Araxá regions, respectively. However, to date, there is no research in the literature on the in vitro probiotic potential of LAB isolated from artisanal cheeses produced in the Serra Geral region, as well as their raw materials and production environment. Thus, the present study aimed to evaluate the in vitro probiotic potential of LAB isolated from those cheeses, raw milk, endogenous starter cultures and swabs from surfaces. The LAB used (Table 1) were previously isolated and identified by Caldeira (2021).
The spot-on-the-law antagonism was performed in duplicate, with three repetitions, using the technique proposed by Tagg et al. (1976). As revealing microorganisms, four pathogens were used, namely: Staphylococcus aureus (ATCC 33591), Salmonella Typhimurium (ATCC 14028), Listeria monocytogenes (ATTC 15313) and Escherichia coli (ATTC 25922); in addition to a LAB (Levilactobacillus brevis), isolated by Valente (2021), from Minas artisanal cheese. For this analysis, the tested LAB were activated twice in MRS broth, incubated at 37°C for 24 h under aerobic conditions. Five μL of the activated culture were placed in the center of Petri dishes containing MRS agar. These plates were incubated at 37ºC for 48h, under aerobic conditions, forming the spots. Afterwards, 1 mL of chloroform was added to the lids of the plates, which were also exposed to ultraviolet light for The artificial gastric acid resistance was performed in duplicate, with three repetitions, using the adapted technique described by Silva et al. (2013). LAB were activated twice in MRS broth, which was incubated at 37°C for 24 h under aerobic conditions. Afterwards, they were centrifuged at 13,000G for 5min. The supernatant was discarded, and the pellets were washed with saline three times. Then, they were suspended in 1 mL of acid solution (hydrochloric acid, pepsin, and distilled water), pH 2.0 -for the experimental group and 1mL of saline solution (NaCl 0.9%), pH 7.0for group control. The samples were incubated at 37ºC for 2 h. The bacteria were centrifuged again 13,000G/5 min and washed with 0.9% saline solution to remove the acid solution, and then the pellets were suspended in MRS broth. A 2% (v/v) inoculum was also prepared in MRS broth. Then, 200µl of each sample from the control and experimental groups were transferred to a 96 well-microplate, which was incubated for 24 h in spectrophotometer equipment (Microplate, Spectrophotometer System 47 SpectraMax 340 -Molecular Devices, Sunnyvale, California, United States) at 37ºC. The absorbance of the material was determined by measuring the optical density (OD) at 620nm every hour, for 24 h, to observe inhibition. Inhibition percentage was calculated according to the formula: (1-SG/AT) x 100, in which SG and AT correspond to the areas under the growth curve of the bacteria treated with artificial gastric acid and the control, respectively. The interpretation of the results was performed based on the criteria proposed by Acurcio et al. (2014). The LAB were considered tolerant when they presented a percentage of inhibition below 40%, moderately tolerant from 40 to 80% and sensitive higher than 80%.
The bile salt resistance was performed similarly to the former test, except that there was no previous incubation of the samples. A volume of 200 µL of the samples treated with 0.3% Oxgall (bile salts) and MRS (control) were placed in the 96-well microplate. Absorbance was determined by measuring the OD 620nm every hour for 24 h, and the percentage of inhibition was calculated using the formula (1-SB/CT) x 100, in which SB and CT correspond to the area under the growth curve of the bacteria treated with bile salts and the control, respectively. The resistance levels were the same described for the former test.
After carrying out the in vitro tests, two BAL that obtained the best results were selected for the elaboration of fermented milks, which were submitted to physical-chemical and microbiological analyses. For this, three different batches of Molico (Nestlé®, Araçatuba, São Paulo, Brazil) skimmed milk powder were used. They were reconstituted with distilled water and sterilized at 110ºC for 10 min.
The selected BAL were evaluated, individually or in association (LLSG + LB), for their fermentative capacity, based on the evaluation of milk coagulation. For this, they were activated twice in MRS broth, which was incubated at 37°C for 24 h under aerobic conditions. After this procedure, 0.25mL of each individual LAB and 0.125mL of each mixed culture were transferred to test tubes containing 10 mL of reconstituted and sterilized powdered milk. They were incubated at 37ºC for 24 h. After that, coagulation was evaluated. Only LAB capable of fermenting milk and producing a firm clot were selected to produce fermented milks.
The fermented milk with the selected LAB was prepared using skimmed milk powder (Molico, Nestlé®, Araçatuba, São Paulo, Brazil) from three different batches, consisting of three repetitions. The skimmed milk powder was reconstituted at 10% in distilled water and was subjected to heat treatment at 110ºC for 10 minutes. They were stored at 8ºC and analyzed after 1, 7 and 14 days and submitted to physical chemical analyzes: determinations of pH (Gehaka PG1800, São Paulo, Brazil), titratable acidity (Official…, 2019); and contents of moisture, protein, and fat (Official…, 2019). The counting of BAL present in the initial inoculums and in fermented milk was also determined at 1, 7 and 14 days of storage, using serial decimal dilutions in saline solution (0.9% NaCl). Afterwards, two 10μL microdrops of the dilutions were placed on MRS agar. The plates were incubated under aerobic conditions at 37°C for 48 h. The counts of microorganisms were made by multiplying the mean number of colonies formed in the two drops by 100 and by the inverse of the dilution (Yogurt…, 1988). Graphpad Prism 7.0 software (Graphpad Software, San Diego, California, USA) was used to perform the statistical analyses. The means of the inhibition halos of the spot-on-the-lawn antagonism were submitted to the analysis of normality by the Shapiro-Wilk test and analysis of variance by the Two-way ANOVA test. The comparison between the means was performed using the Kruskall-Wällis test with a significance level of 5%. The physicochemical parameters of the fermented milks were subjected to normality analysis using the Shapiro-Wilk test, the Twoway ANOVA and means were compared using the Tukey test, with a significance level of 5%. For bacterial count evaluations, the same statistical analysis was used after logarithmic transformation of the data.
The results of the susceptibility of the tested LAB to antimicrobials of clinical importance are shown in Table 2.   It was possible to observe that all evaluated LAB were sensitive to penicillin, tetracycline, and chloramphenicol. Similar results were found by Valente et al. (2019) when evaluating the sensitivity profile of Lactobacillus to antimicrobials. Susceptibility to chloramphenicol was already expected, as its use in livestock is prohibited, due to the side effects on the bone marrow of the humans (Stival et al., 2021).
According to the World Health Organization (WHO), antibiotic resistance is rising to dangerously high levels in all parts of the world as new resistance mechanisms are emerging and spreading globally. With antibiotics becoming less effective, it has grown increasingly difficult to treat patients for even common infectious diseases like pneumonia (Antibiotic…, 2020).
A total of 87.5% of the LAB showed resistance to vancomycin. Similar results were found in other studies (Costa et al., 2013;Andrade, et al., 2014;Valente et al., 2019) with LAB isolated from Minas artisanal cheeses. Most LAB (87.5%) showed resistance to oxacillin. The same was reported by Silva et al. (2019), when evaluating the probiotic potential of Lactobacillus spp. isolated from Minas artisanal cheeses from the Araxá region.
Most of the LAB (75%) showed resistance to ciprofloxacin, which can be justified due to intrinsic characteristics of these microorganisms, related to cell wall structure, membrane permeability and efflux mechanisms (Abriouel et al., 2015). These results agree with those obtained by Costa et al. (2013) and Andrade et al. (2014).
A total of 75% of the LAB showed resistance to gentamicin and all LAB were resistant to amikacin, possibly due to mechanisms related to membrane permeability or absence of cytochromes that would allow the binding of antimicrobials (Abriouel et al., 2015).
From the results presented in Figure 1, it was possible to observe that all samples were able to produce inhibition halos against the tested pathogens. In addition, no LAB produced halos against the LAB isolated from Minas artisanal cheese, which is desirable for LAB screening. Different results were found by Costa et al. (2013) and Valente et al. (2019), who described tolerant LAB isolated from Minas artisanal cheeses to acid environment. Thus, the selected LAB in the present study should be encapsulated, to survive the acid of human stomach and reach the intestine in concentrations needed to show desirable effects in the host's gastrointestinal tract.
In addition, selected potential probiotics can be conveyed into a dairy derivative since food products, such as dairy, can contribute to the survival of probiotics in gastric juice, mainly due to their buffering and protective effect (Silva et al., 2021).
Regarding bile salt resistance, 87.5% of the LAB (7/8) were tolerant, only one was moderately tolerant and none were sensitive to this adverse condition. Possibly due to the development of defense mechanisms, such as the deconjugation of bile salts by the enzyme bile salt hydrolase (BSH) (Peres et al., 2014). These findings differ from the results found by some researchers, in which most of the potential probiotic LAB proved to be sensitive or moderately tolerant to bile salts (Costa et al., 2013;Valente et al., 2019).

Lactococcus lactis (LLSG) and
Levilactobacillus brevis (LB) were selected to produce fermented milk, as they showed the best results in vitro (Table 4). In addition, Lactococcus lactis is the main LAB species used as a starter culture for several foods, being commonly used by industries for fermented dairy products manufacture, such as cheeses. This culture plays a key role in relation to the quality of these products, their shelf life, preservation, and sensory attributes (Pereira et al., 2020).     After the fermentation test, only milk inoculated with LLSG and association of LLSG and LB produced a firm clot. As a result, only LLSG was selected for the elaboration of fermented milk.

S t a p h y l o c o c c u s a u r e u s
The search for pathogens in reconstituted milk powder showed the absence of Salmonella spp. and undetectable counts of coliforms (NMP/mL), Staphylococcus spp., molds, and yeasts, demonstrating that the product was adequate for the elaboration of fermented milks. The inoculum used to prepare fermented milk had a count of 2x10 8 CFU/g LAB. LAB counts in fermented milks remained high (Fig. 2) and in accordance with current legislation (Brasil, 2007) throughout the storage period.
Regarding the physicochemical analysis performed on milk fermented with LLSG at 1, 7 and 14 days of storage at 7ºC (Fig. 3), it was possible to observe a gradual increase in its acidity throughout the storage period. The acidity values were according to the current legislation (Brasil, 2007). With the increase in acidity, a decrease in the pH of the medium was expected. Despite this, it was possible to observe a gradual increase in this pH. In contrast to the production of acids, it is possible that the formation of alkaline compounds had occurred, probably on a larger scale, causing the pH to increase.
The most likely hypothesis for this formation of alkaline compounds is due to proteolysis, related to the action of proteases present in the milk or produced by LAB (Freire et al., 2021). This finding supports this hypothesis since reduction in the protein content was observed during the storage period. The protein content was slightly below the minimum recommended by the legislation (Brasil, 2007) on days 1 and 14 of storage.
High moisture content was observed in the fermented milk, varying from 90.98 to 89.71 during storage. In relation to the fat content, undetectable values were observed during the entire period of storage since skimmed milk powder was used for the preparation of fermented milk. This value is in accordance with the standards established in the Brazilian legislation (Brasil, 2007).
From the results presented, it is suggested that LLSG is suitable to be used as a potential probiotic to obtain fermented milk. However, it is necessary to carry out in vivo screening tests for its future use as a probiotic and elaboration of functional foods. Also, the present study showed the presence of beneficial bacteria in the microbiota of artisanal cheeses produced in the Serra Geral region. Figure 2. LAB count in fermented milk at 1, 7 and 14 days of storage at 8ºC.