Partial characterization of probiotic lactic acid bacteria isolated from Chinese dairy products

ABSTRACT A total of 246 bacterial isolates were collected from various food and animal sources within Nanning city, China. Sequencing of the 16S rRNA gene revealed that 24.4% of the isolates were lactic acid bacteria (LAB). Initial screens identified three isolates from semi-hard cheese: Lactobacillus plantarum subsp. plantarum strains K3 and K4 and Lb. paracasei subsp. tolerans K8; and one from raw buffalo milk: Lb. plantarum subsp. plantarum E41 as having the highest levels of cell surface hydrophobicity and auto-aggregation. Further characterization of these strains showed that all four isolates were completely stable after exposure to the pepsin and trypsin for 6 hours, and strains K8 and E41 remained viable after 6 hours of exposure to acidic conditions (pH 2.5) and bile salts (0.3%). In addition, strains K3 and K4 were shown to inhibit the growth of potential human pathogens, including methicillin resistant Staphylococcus aureus, Escherichia coli and Salmonella typhi, with inhibition zones being >14 mm for each bacterial target. Results from this study suggested that the four characterized LAB strains could survive passage through the gastrointestinal tract, thus supporting the need for additional studies to assess their potential as probiotics.


INTRODUCTION
The protective role of a host's indigenous microbiome against colonization of the gastrointestinal tract by foreign, potentially pathogenic, microorganisms has been considered the scientific basis for the development of probiotics. [1] In 2001, the joint Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) Working Group defined probiotics as "live microorganisms which when administered in adequate amounts confer a health benefit on the host." [2] In 2014, the International Scientific Association for Probiotics and Prebiotics retained the FAO/WHO definition with a minor grammatical correction as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." [3] Several bacterial and fungal species have been investigated as potential probiotics, including species of lactic acid bacteria (LAB) and bifidobacteria. A variety of potential health-promoting activities have been attributed to the use of probiotics including: alleviation of inflammatory bowel disease, food allergies, lactose intolerance and hypercholesterolemia; anticancer, immunoregulatory and antioxidant effects; and the prevention of bacterial pathogen colonization and proliferation. [4] Since these bioactivities are often strain dependent, at 37°C for 48 h. After incubation, the plates were observed for characteristic hemolytic zones (α, β or γ hemolysis) . [9] Cell surface hydrophobicity assay LAB isolates were cultured in MRS broth for 24 h at 37°C. Cells were collected by centrifugation (4000 x g for 10 min), washed twice with sterile saline (pH 7.0), and resuspended in the same solution at an OD 600 of 0.6-0.7 using a UV/Vis spectrophotometer (Metash UV-800, Shanghai, China). Two milliliters of the bacterial suspension were mixed with 2 mL of xylene and vortexed for 2 min. The mixture was then left undisturbed for 30 min to allow the two phases to separate, [10] and the cell surface hydrophobicity (CSH) was calculated as a percentage from three replicates according to the following equation: CSH% = [(A 0 -A)/A 0 ] × 100 where A 0 is the OD 600 before mixing and A is the OD 600 after 30 min of mixing.

Auto-aggregation assay
LAB broth cultures were centrifuged at 4000 x g for 10 min, and the harvested cells were washed twice and re-suspended in saline solution (OD 600 0.6-0.7). Suspensions were incubated at 37°C for 1, 2, 3, 4 and 5 hours, after which the auto-aggregation (AA) percentage was calculated from four replicates using the following equation [10] : where A 0 and A t represent OD 600 at zero time and OD 600 after selected time of incubation, respectively.

Gastrointestinal tolerance
The tolerance of LAB to conditions and enzymes within the gastrointestinal tract was assessed through exposure to bile salt (0.3%), acidic pH (2.5), pepsin (5 mg/mL) and trypsin (10 mg/mL), separately. For all tests, LAB were collected from an overnight broth culture by centrifugation at 4000 x g for 10 min, washed twice with saline solution and inoculated (10 7 -10 8 cfu/mL) into MRS broth supplemented with bile salt, pepsin or trypsin. Additionally, LAB were resuspended in MRS broth with an adjusted pH to 2.5 to test for acid tolerance. [11] LAB suspensions were incubated at 37°C, with viable cell counts determined using the pour-plate method after 2, 4 and 6 hours.

Antibacterial activity assay
The antibacterial activity of LAB isolates was assessed against methicillin resistant Staphylococcus aureus (MRSA) ATCC 43300, Escherichia coli ATCC 25922 and Salmonella typhi ATCC 19430 using a well diffusion assay (cup -plate method). [12] LAB were grown in MRS broth at 37°C for 24 h, after which cells were removed by centrifugation at 8000 x g for 10 min. The cell-free supernatant (CFS) was filtered through a cellulose acetate membrane filter with a pore size of 0.45 µm. The cup -plate method was performed using 3 sterilized Oxford cups placed equidistant on an agar base medium (2% agar). Subsequently, a layer of nutrient agar inoculated with 1% (v/v) of the indicator bacterial strain was overlaid on the agar base. Once solidified, all cups were removed, and the wells were filled with 150 µL of CFS. Plates were incubated at 37°C for 24 h and observed for the appearance of clear zone around the wells. The diameter of each inhibition zone (mm) was measured using vernier caliper and the results were expressed as mean ± SD. Measurement of cell-free supernatant pH was performed in parallel with this experiment using pH meter (PB-10, Sartorius Stedim Biotech GmbH, Gottingen, Germany).

Growth kinetics and acid production
To evaluate the relationship between LAB growth and acid production, MRS broth (150 mL) with an initial pH of 5.3-5.8 was inoculated with 2% (v/v, 3 mL) of an overnight MRS culture for each LAB isolate. The initial optical density was 0.6 at 600 nm to standardize the bacterial count to approximately 10 8 cfu/mL. The growth curve was established by monitoring the optical density (OD 600) every 2 hours for 24 hours. At each time point, the pH value was also measured using pH meter (PB-10). The growth rate and generation time were calculated applying the following equations [13] : Growth rate (r) = [ln (OD 2 /OD 1 )]/T 2 -T 1 As OD 1 and OD 2 are the OD 600 values at the time T 1 and T 2 , respectively. Generation time = ln2/r

Statistical analysis
The data were presented as mean ± standard deviations (SD). One-way analysis of variance (ANOVA) was performed by Statistix 8.1 (Analytical Software, Tallahassee, USA) followed by assessment of differences by Tukey's test at P < .05.

Auto-aggregation assay
The 10 isolates with the highest CSH were further tested for the auto-aggregation (AA) capability (Table  2). Results showed that auto-aggregation increased over time for most strains, with the exceptions being two Lb. fermentum isolates (K41 and K43) and Lactococcus petauri isolate L22, where AA% peaked at 4 h ( Table 2). Three Lb. plantarum subsp. plantarum isolates (K3, K4 and E41) and one Lactobacillus paracasei subsp. tolerans isolate (K8) showed optimal CSH and intermediate to optimal AA activity. Lactobacillus plantarum subsp. plantarum K3 and K4 showed the highest levels of CSH and AA, and the variation in their observed levels suggested they were two novel strains of this species. Based on the CSH and AA results, Lb. plantarum subsp. plantarum isolates K3, K4 and E41; and Lactobacillus paracasei subsp. tolerans K8 were selected for further characterization of potential probiotic traits.

Gastrointestinal juice tolerance
Lb. plantarum subsp. plantarum isolates K3, K4 and E41, and Lactobacillus paracasei subsp. tolerans K8 were further evaluated for their ability to survive exposure to stresses present within the GIT, Data are means ± SD (n = 3). Different superscript letters in the same column are significantly different (P ˂ 0.05).

Lactic acid bacterial growth and acid production
Growth and acid production were monitored for the four LAB isolates in MRS broth for 24 hours (Figure 1).  toleransK8 had a growth rate of 0.7 h and a generation time of 1.0 h. The culture pH values were also recorded after 13 h and dropped from the initial 5. 8-5.9 to between 4.1 and 4.5 by the end of exponential growth (Figure 1), which agreed with results reported in Table 4.

DISCUSSION
Extensive research has focused on the isolation and characterization of probiotics from human sources, but recent investigations have emphasized the characterization and identification of probiotic bacteria isolated from other sources such as fermented foods and dairy products. [14] In this study, 60 LAB isolates were obtained from either raw buffalo milk, semi-hard cheese, smoked cheese, yogurt, pickled vegetables, fermented forage and swabs obtained from the area surrounding the buffalo udder. All isolates were identified using 16S rRNA gene sequencing and shown to be gamma-or nonhemolytic, suggesting they were safe to pursue as potential probiotic strains. All LAB isolates were evaluated for cell surface hydrophobicity (CSH), with the 10 strains showing the highest CSH activity further screened for their auto-aggregation (AA) ability. CSH and auto-aggregation (AA) are potentially important traits for probiotic candidates, as they would affect the bacterium's ability to adhere to the intestinal mucosa and form biofilms on different surfaces. [15] Previous studies have suggested cells with a CSH > 40% are considered hydrophobic . [16][17][18] In the current study, 80% of tested LAB isolates had a CSH between 47.54 ± 6 and 97.13 ± 0.8%, confirming their hydrophobic nature. Furthermore, seven LAB isolates with the highest CSH activities also showed auto-aggregation levels above 25%. These results are comparable to previous studies which showed LAB with comparable CSH and AA activities survived exposure to gastrointestinal stresses and adhered to epithelial cells. [17][18][19] These activities are believed to increase the probiotic potential of LAB allowing them to persist longer within the host to convey their health benefits. [20] CSH and AA results from this study suggested that isolates K3, K4 and E41, identified as novel strains of Lb. plantarum subsp. plantarum, and isolate K8, identified as Lb. paracasei subsp. tolerans, should be further investigated for their potential as probiotics.
In order to convey a health benefit to the host, probiotic bacteria must be able to survive passage through the GIT [17,[21][22][23][24] ; and results from this study showed that Lb. plantarum subsp. plantarum isolates K3, K4, and E41; and Lb. paracasei subsp. tolerans K8 showed little or no loss in cell viability after in vitro exposure to low pH and digestive enzymes (pepsin and trypsin). Resistance to bile salts has also been reported to be one of the most essential properties required of a potential probiotic active within the small intestine [17] ; and in this study, Lb. plantarum subsp. plantarum E41 and Lb. paracasei subsp. tolerans K8 were resistant to bile salt exposure for up to 6 h. Viability of Lb. plantarum subsp. plantarum isolates K3 and K4 was reduced by 2-3 log CFU/ml after 6 hours of exposure to bile salts. This loss in viability was most likely due to leakage of intracellular material as bile salts have been reported to permeabilize bacterial cell membranes. [25] Previous studies have shown that the antimicrobial activity of bile salts depends on their concentration. High concentrations have been reported to dissolve membrane lipids, causing leakage of cell materials and cell death, while low concentrations may affect membrane fluidity and permeability by altering membrane-bound proteins or increasing the flow of divalent cations through the membrane. [26,27] Our results suggest that the Lb. plantarum subsp. plantarum isolates K3 and K4 may require protection when being delivered if they are intended to reach the colon to convey health benefits to a host.
Another probiotic trait investigated was the inhibition of human pathogens by Lb. plantarum subsp. plantarum isolates K3, K4 and E41; and Lb. paracasei subsp. tolerans K8. LAB have been reported to display antagonistic activities against pathogenic and food spoilage microorganisms through production of lactic acid, or other antimicrobial agents including carbon dioxide, hydrogen peroxide and low molecular peptides, called bacteriocins. [28] The antimicrobial effect of H 2 O 2 may result from oxidation of sulfhydryl groups causing denaturation of enzymes, and from the peroxidation of membrane lipids causing permeabilization of the cell membrane. H 2 O 2 may also be a precursor for the production of bactericidal free radicals which can damage DNA. [29] Carbon dioxide is mainly produced by heterofermentative LAB and contributes to the creation of an anaerobic environment which inhibits enzymatic decarboxylation. In addition, accumulation of CO 2 in the lipid bilayer may cause dysfunction of the cell membrane. [30] Bacteriocins are peptides or peptide complexes (usually 30-60 amino acids) naturally produced by some LAB, which exhibit bacteriostatic or bactericidal activity mainly against closely related species. [31] However, some bacteriocins also display broad-spectrum activity, which has resulted in their investigation as alternatives to chemical food preservatives or antibiotics. [29] While several mechanisms of action have been shown for these peptides, many act by forming pores within the bacterial cell membrane, resulting in the leakage of intracellular material and possibly cell death. [32] In this study, cellfree supernatants from Lb. plantarum subsp. plantarum K3 and K4; and Lb. paracasei subsp. tolerans K8 were shown to have antimicrobial activity against MRSA, E. coli and Salmonella typhi. Recent studies reported the antimicrobial activity of Lb. plantarum against Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes. [33,34] While there are some reports describing LAB bacteriocin activity against Gram-negative foodborne pathogens, [35] these peptides are more often active against Gram-positive pathogens, such as Listeria monocytogenes. [36] More studies are required to determine the antimicrobial compounds produced by these three LAB isolates, but the lack of antimicrobial activity for CFS from isolate E41, which had a similar pH to the others, suggests that the production of organic acid alone is not responsible for the broad-spectrum activity.
In addition to surviving passage through the gastrointestinal tract, LAB that are intended to serve as probiotics within food must be able to survive under typical storage conditions for the intended product. In this study, Lb. plantarum subsp. plantarum isolates K3, K4, E41, and Lb. paracasei subsp. tolerans K8 were all shown to survive at refrigeration temperature (4°C) without a reduction in viable cell count after 15 days of storage. This suggested that they may be ideal cultures for use in fermented dairy products which are stored under refrigeration. Future studies are needed to investigate if the cultures are capable of surviving at this temperature within a dairy environment.
Finally, the relationship between growth and acid production was assessed for Lb. plantarum subsp. plantarum isolates K3, K4, and E41, and Lb. paracasei subsp. tolerans K8. As expected, the greatest drop in pH occurred during the log phase for all isolates as the pH was reduced by 28.4, 27.6, 23.7 and 28.6% for each isolate, respectively. The final pH for all cultures was ≤4.2, which was similar to previous reports for fermented meat products where LAB cell counts reached 6-8 log cfu/g of meat. [37][38][39]

CONCLUSION
In conclusion, the Lactobacillus plantarum subsp. plantarum isolates K3, K4 and E41, and Lactobacillus paracasei subsp. tolerans K8 were partially characterized in vitro to demonstrate their potential probiotic traits. Results from these studies suggest that Lb. paracasei subsp. tolerans, which was isolated from semi-hard cheese, exhibited promising probiotic properties, including antimicrobial activity against MRSA, E. coli and Salmonella typhi. This isolate also had high cell surface hydrophobicity and auto-aggragation capability; and cell viability was not affected by the gastrointestinal stresses studied. Lb. plantarum subsp. plantarum E41, isolated from buffalo milk, was also resistant to the gastrointestinal stresses; however, it did not show antimicrobial activity against any of the targeted pathogens. Lb. plantarum subsp. plantarum isolates K3 and K4 were negatively affected by exposure to bile salts but were resistant to the other GIT stresses tested and displayed broad-spectrum antimicrobial activity against the targeted bacterial pathogens. The results presented in this study are promising for the probiotic potential of these four strains; more studies are required to assess their true potential, including antibiotic susceptibility, co-aggregation capability with pathogenic bacteria, simulated gastrointestinal juice tolerance, and phenol tolerance.