Isolation and Characterization of Lactobacillus crispatus, Lactococcus lactis, and Carnobacterium divergens as Potential Probiotic Bacteria from Fermented Black and Green Olives (Olea europaea): An Exploratory Study

Background Table olives are becoming well recognized as a source of probiotic bacteria that might be used to create a health-promoting fermented food product by traditional procedures based on the activities of indigenous microbial consortia present in local environments. Methodology. In the present study, the characterization of probiotic bacteria isolated from mince, chunks, and brine of fermented green and black olives (Olea europaea) was done based on morphological, biochemical, and physiological characteristics. Results Bacterial isolates demonstrated excellent survival abilities at 25, 37, and 45°C and at a variable range of pH. However, the optimum temperature is 37 and the optimum pH is 7 for all three isolates. An antimicrobial susceptibility pattern was found among these isolates through the disc diffusion method. Most of the isolates were susceptible to streptomycin, imipenem, and chloramphenicol, whereas, amoxicillin showed resistance to these isolates, and variable results were recorded for the rest of the antibiotics tested. The growth of the isolates was optimum with the supplementation of 3% NaCl and 0.3% bile salt. The isolated bacteria were able to ferment skimmed milk into yogurt, hence making it capable of producing organic acid. Conclusion Isolates of Lactobacillus crispatus MB417, Lactococcus lactis MB418 from black olives, and Carnobacterium divergens MB421 from green olives were characterized as potential candidates for use as starter cultures to induce fermentation of other probiotic food products.


Introduction
Probiotics have a long history of human consumption; for example, cultured dairy products (curds and yogurts) are traditionally consumed in several parts of the world. The word probiotics is of Greek origin, meaning "for life", which is the antonym of antibiotics [1,2]. Those living microorganisms that are administered in an adequate amount and have a beneficial effect on human health are known as probiotics [3,4].
According to Ranadheera et al., [5] and Oleskin, & Shenderov [6], the human gastrointestinal tract contains more bacteria than eukaryotic cells. This gut flora also contains probiotics, which constructively influence the body by offering health encouragement to the host [4].
Microorganisms used as probiotics, specifically lactic acid bacteria (LAB), include Lactobacillus species, some species of Bifidobacteria, Enterococcus, and Streptococcus [7,8]. Most of these bacterial species reside in the human intestine [9]. The only probiotic yeast that exists is the nonpathogenic Saccharomyces boulardii [10][11][12]. LAB has prevalent exploitation in fermented food manufacturing [13] and is a generally recognized as safe (GRAS) organism that can be securely used for medical or veterinary purposes [14,15].
If LAB is exposed to traumatic circumstances in the gastrointestinal tract like acidic gastric juice, bile salt, and/or altered microbial balance of the intestinal tract, then administration of any kind of antibiotic may suppress the probiotic population. So, to get the maximum benefits out of the probiotic bacterial population, the selection of probiotics is made after various in vitro and in vivo tests [16,17].
There are a variety of traditional fermented food products produced by probiotic fermentation like fruits and vegetables including olives, beetroot, cabbage, and other leafy vegetables. Fermentation is done by keeping the vegetables in a 2% brine solution and allowing the stored vegetables to be fermented with LAB [18,19]. In the food industry, especially for ready-to-eat foods, microbiological quality is a persistent concern. Lactic acid bacteria (LAB) could be used to prevent the growth of spoiling and pathogenic bacteria [20]. Some LAB exhibits active microbial antagonism through competition mechanisms, the production of bacteriocins, or the production of organic acids [21][22][23].
The plant named "olive" is a small tree of the family Oleaceae, and its binomial name is Olea europaea [24,25]. Olives are bitter when raw or fresh, so they must be treated and fermented to make them edible. Both green olives, which are full-size olives plucked before ripening, and black olives, which are completely matured, ripened olives, can be fermented. The main reasons for olive processing are the exclusion of bitterness by hydrolysis of some phenolic compounds (like oleuropein), preservation of the fruit, and enhancement of the organoleptic characteristics of the ultimate product [26,27].
Due to the high concentration of dietary fiber, vitamins, antioxidants, and anticancer chemicals in table olives, they have therefore been considered useful food [28]. Table olives are called pickled vegetables, in which preparation and maintenance are achieved by an amalgamation of salting, fermentation, and acidification. Some studies have been conducted to widen the range of useful food types by utilizing of the microarchitecture of the surface of olives, and the dietary characteristics of olive pulp to produce a flavorsome, vegetable-based efficient food comprising of table olives equipped with probiotic strains [29].
In research, the capacity of seven strains from the probiotic species Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium umbifidum, and Bifidobacterium longum to survive on the olive surface and the suitability of table olives as a biological carrier for probiotic microorganisms were studied [30]. The resultant table olives can be stored with or without refrigeration, and probiotic-dominated fermentations are generally considered the most suitable method of curing olives [31,32]. This study is aimed at identifying and functionalizing the characterization of isolated probiotics from commercially available green and black olives.

Materials and Methods
The research work was done in the Microbiology & Biotechnology Laboratory at Fatima Jinnah Women University, Rawalpindi. The work is divided into two phases: isolation and identification of bacteria and physiological characterization of probiotic isolates.

Sample Collection.
Fermented pitted Spanish green and black olive samples of the same brand (Figaro Company) were purchased from local superstores located in the Pakistan Aeronautical Complex (PAC), Karma, and Rawalpindi. The sealed fermented green and black olives bottles were opened in sterile condition near a flame to avoid contamination. Using sterile forceps, a few olives from the bottles were taken out and then minced inside a mortar and pestle after disinfecting it with an ethanol (70%) swab. Before being minced, big chunks of both black and green olives were kept inside sterile universal bottles with the help of sterile forceps; later, the olive mince from the mortar was also collected in universal bottles and infused in autoclaved distilled water using a sterile spatula. The brine from black and green olive samples was also stored in a sterile universal bottle following  [34,35]. Isolations from chunks, brine, and minced fermented black and green olives were done on MRS media by the spread plate method [36]. Isolates were characterized by morphological, biochemical, and physiological characteristics.

Morphological and Biochemical
Characterizations. Morphological characterization was studied through the gram staining technique by Hans Christian Gram [37]. The catalase test [38], Simmons citrate test [39,40], methyl red and Voges-Proskauer's test [41], indole production test [40,42], and oxidation fermentation test [43,44] were performed for biochemical characterization of the isolates. The identification of the isolated bacteria was done by the Standard API 50-CHL system [45].

Physiological Characterization
2.4.1. Determination of Optimal Temperature. Isolated bacteria were incubated at 25°C, 37°C, and 45°C for 24 hours to determine their optimal temperature for growth, and the results were calculated by the spectrometric method at 600 nm [46].

2.4.2.
Determination of Optimal pH. Fresh bacterial cells were grown for 24 hours at pH ranges of 4-9 to determine whether the isolates were acidophilic, neutrophilic, or alkaliphilic. Optical density was measured by spectroscopic readings at 600 nm [46].

The Antibiotic Sensitivity
Assay. An antibiotic susceptibility test was conducted using the disc diffusion method [47]. Fresh overnight cultures of bacterial isolates were spread onto Mueller-Hinton (MH) agar media plates (recommended by the National Committee for Clinical

Antimicrobial Activity.
The antimicrobial activity of all isolates against indicator bacteria was determined by the agar-well diffusion method [48]. Pseudomonas geniculate, Microbacterium oxydans, Bacillus subtilis, Streptomyces laurentii, Klebiella pneumonia, Bacillus pumilus, Bacillus cereus, Acaligens facelis, Enterococcus faecium, and Enterococcus facelis were used as indicator bacteria (obtained from the Microbiology & Biotechnology Lab, FJWU) against which the antimicrobial activity of isolated strains was assayed. Each test string was inoculated into 5 ml of nutrient broth and incubated at 37°C for 24 hours on a shaking incubator at 150 rpm. After incubation, each culture was centrifuged at 10000 rpm for 5 minutes to obtain a cell-free supernatant. Supernatants of isolated species were identified for antibacterial activity besides indicator bacteria. For antimicrobial activity identification, Mueller-Hinton agar medium (for antimicrobial testing) was prepared, autoclaved, and poured separately into the sterile Petri dishes. By the spread plate method, the plates were inoculated with indicator bacterial suspension. Five wells, each with an 8 mm diameter, were generated in every nutrient agar plate, and the base of each well was sealed with soft agar (0.7%) plugs. To identify the antibacterial activity of probiotic isolates, 100 μl of cell-free supernatants of probiotic strain was added to the well. The plates were incubated for 24 h at 37°C, and the diameter of the zone of inhibition was measured in millimeters on both nutrient agar and MH agar media.
+Gram positive: S F: spore formatio; Cat: catalase production; M R: methyl red; V P: Voges-Proskauer; I P: indole production; S C: Simmon's citrate; A P: acid production. 2.4.6. Organic Acid Production Assay. Lactic acid bacteria are known for the production of organic acids, specifically lactic acids. Isolates were considered to have the same property, and to determine this ability, an acid production assay was conducted [14]. The brine in which fermented green olives and black olives were stored was also assayed for organic acid produced by the isolated strains inhabiting the brine at the beginning, in the middle, and at the end of the research work. Powdered skim milk was purchased from the local market. Autoclaved distilled water was taken and mixed with 10% of powdered skim milk to make sterile skim milk with a pH of 6.68. Five ml of skimmed milk was inoculated with a 24 h fresh culture of isolated bacteria and incubated at 37°C for 24, 48, and 72 h. After incubation, coagulated skim milk was filtered, and the pH of each filtrate was measured with a digital electrode pH meter for lactic acid production. The filtrate was also titrated with 0.1 N NaOH, and organic acid production was quantified in terms of percentage strength.

MB421
2.4.7. Bile Salt Tolerance Assay. The ability of isolated species to survive and grow in the presence of bile salts was investigated by growing the isolated strains with different bile salt concentrations (0.1, 0.3, 0.5, and 1%) in nutrient broth as described by Dunne et al. [47]. The broth was incubated for 4 h at 37°C, and the optical density (OD) of the cultures was then measured at 600 nm. The viability and growth of isolates at this concentration showed tolerance of isolated strains within the gastrointestinal tract of humans [51,52].
2.5. Tolerance to Simulated Gastric Juice. Stimulated gastric juice tolerance was determined by the method described by Graciela and Maria [52]. Stimulated gastric juice was freshly prepared and sterilized by filter sterilization.
2.6. Numerical Taxonomy of Probiotic Isolates. Similarity among isolates was checked by taking data from fifty (50) different biochemical sugar fermentation tests from the API CH 50 kit and converting them into binary data (0 or 1) for negative or positive test results, respectively, using PAST (Paleontological Statistics Software Package for Education and Data Analysis software). Similarities amongst the strains were estimated using the Jacquard coefficient, and the unweighted average linkage gave the cluster.

Isolation and Identification of Potential Probiotic
Bacteria. In the present study, probiotics were isolated from commercially available fermented black and green olives from the Figaro Company. Fermenter probiotic bacteria were isolated on an MRS medium, and a total of 12 isolates were obtained from brine, chunks, and suspension from minced olives ( Figure 1). As shown in Table 1, potential probiotic bacteria were isolated from black and green olives. Identification was done by cell and colony morphology, which showed diversity ranging from 0.5 to 11.5 mm in size, circular to irregular shape, and white to pale color. All the isolated probiotic bacteria were stained as Gram-positive, and most of them were rod-shaped. For Lactobacillus crispatus, Lactococcus lactis, and Carnobacterium divergens, the results of biochemical tests showed characteristics similar to those of reported 5 BioMed Research International probiotics, for example, not being catalase producers (anaerobes or facultative anaerobes that only ferment and do not respire using oxygen as a terminal electron acceptor), being nonspore formers, and having a thick peptidoglycan cell wall structure (gram-positive). The isolates ferment glucose during anaerobic incubation as well as during aerobic incubation, but no gas was produced during the 24 h of incubation, both under aerobic and anaerobic conditions ( Table 2). Whereas the isolates were identified by carbohydrate fermentation patterns on the API 50 CH panel, the carbohydrate fermentation ability of the isolated strains was analyzed against frothy nine different sugars ( Figure 2). The heat map summarizes the sugar fermentation pattern of probiotic bacteria from fermented olives and groups the strains based on similarities and differences in the sugar fermentation profile shown in Figure 3. The species-level identification was done using API web50 CHL v5.1 (Table 3).

Characterization of Optimal
Temperature and pH. Carnobacterium divergens MB421 from green olives and Lactoccocus lactis MB418 from black olives grew optimally at 25°C, and Lactobacillus crispatus MB417 showed optimal growth at 37°C (Figure 4). Optimal growth of Carnobacterium divergens MB421, Lactobacillus crispatus MB417, and Lactoccocus lactis MB418 was observed at pH 7, showing the neutrophilic behavior of the isolates ( Figure 5).

Evaluation of Antibiotic Susceptibility
Profile. The antibiotic susceptibility of isolates to various antibiotic discs by disc diffusion assay was determined in terms of standard inhibitory zones, and the results are shown in Table 4. All the isolated strains were sensitive to imipenem. Lactobacillus crispatus MB417 and Carnobacterium divergens MB421 showed resistance to amoxicillin.
On Mueller-Hinton (MH) agar medium, isolates behaved differently as compared to nutrient agar medium. This might be due to the MH medium, which is the standard medium for the performance of such tests in microbiology laboratories. Lactobacillus crispatis MB417 and Lactococcus lactis MB418 isolated from black olives were sensitive to Streptomyces laurentii MB319. Isolates depicted no inhibitory zone against Microbacterium oxydans MB325, Klebsiella pneumoniae MB081, and Acaligens facelis MB090, but   3.5. Bile Salt Tolerance Assay. Isolated probiotics were able to tolerate 0.1-1% bile salt and flourish at 0.3% gastrointestinal bile salt concentration ( Figure 6). The isolates Lactobacillus crispatus MB417 and Lactococcus lactis MB418 grew optimally at 0.3% bile salt concentration with a gradual decrease to higher concentrations that were 0.5% and 1.0%, whereas Carnobacterium divergens MB421 showed a linearly increas-ing growth trend starting from 0.1 to 1% bile salt concentration.
3.6. Tolerance to Simulated Gastric Juice. Potential probiotic bacterial isolates were able to tolerate the acidic pH (2) of stimulated gastric juice through the course of incubation, but some isolates were unable to withstand such harsh conditions in the gut. Colony-forming units per milliliter (CFU/ ml) were calculated for black and green olive isolates (Figure 7). Carnobacterium divergens MB421 showed a gradual reducing trend during incubation inside the artificially stimulated gastric juice; while Lactococcus lactis MB18 showed the same reducing viability trend till 90 min of incubation (might be the acclimatization time). The viability  3.7. Salt Tolerance. Following the assessment of salt tolerance, isolates were shown to be resistant to high NaCl concentrations (Table 5), even at a 9% concentration of salt, especially the black olive isolate Lactobacillus crispatus MB417. Isolates Lactococcus lactis MB418 and Carnobacterium divergens MB421 endure up to 8% and 7% NaCl concentrations, respectively.
3.8. Quantification of Organic Acid Production. All three isolates exhibited the capability to coagulate skim milk and pro-duce organic acid under gradually decreasing pH (Table 6). Against 0.1 M NaOH, Lactobacillus crispatus MB417 produced elevated organic acids during 24 to 72 h. Similar results were shown by Carnobacterium divergens MB421, but in the case of Lactococcus lactis MB418 decreased with time, measured at 24, 48, and 72 h of incubation. The organic acid production ability of residing potential probiotic bacteria inside the brine (in which fermented olives were stored) was assayed during research work, that is, at the beginning (1st stage), in the middle (2nd stage), and at the end of practical work (3rd stage). During incubation, the pH and acidity of the brine remained unchanged; this could be because of the refrigerated storage of the fermented olives (Table 7).

Discussion
The present study was conducted to isolate and characterize the potential probiotic isolates from fermented black and green olives. Table olives are the best source of probiotic bacteria [53]. Table olives are considered functional foods because of their nutritional value related to the presence of phenolic compounds and monounsaturated fatty acids [54].
The oxidation-fermentation test in this study identified that the isolated bacteria as facultative anaerobes about their ability to perform fermentation in aerobic as well as anaerobic conditions. The results indicated that all the isolates were able to conduct fermentation during aerobic conditions. All the isolates ferment lactose to produce acid, which in turn changes the color of the medium from green to yellow, showing the isolates were fermenters. Positive fermentation ability was also reported by other scientists on lactic acid bacteria from table olives [55][56][57] and lactic acid bacterial isolates from yogurt [14].
The study showed that most of the isolates were mesophiles, as they prefer 37°C, but some of the isolates were slightly thermophilic. Isolates from another study on green Algerian olives demonstrated tolerance to temperatures ranging from 15 to 45°C [57,58].
In the case of the isolation of probiotics from yogurt, isolates could tolerate pH up to 2.5 with good growth, but according to the current study, after pH 4, growth was very low. This was most likely because isolates from yogurt or dairy origins are more adapted to low pH than isolates from vegetables and meat (due to the source's lactose content, which is then converted to lactic acid after fermentation) [14,59]. Lactobacillus crispatus MB417, Lactococcus lactis MB418, and Carnobacterium divergens MB421 showed good growth at pH 7, depicting their mesophilic nature. Similarly, mesophilic (pH 6.5-7.5) strains of lactic acid bacteria were reported from various research data [14,[57][58][59].
The genomes of probiotic bacterial isolates may contain many antimicrobial resistance genes, van(X), van(E), gyr(A), and tet(M) genes that code for resistance to the respective vancomycin, ciprofloxacin, and tetracycline antibiotics [60][61][62]. As a key feature, a good probiotic candidate should not possess or acquire any antibiotic-resistance genes. This research found that most of the isolates were resistant to amoxicillin. This might be because of the wide and nonspecific use of antibiotics that could subsidize the propagation of resistance in the bacterial population used for the fermentation of olives [63]. Along with this, in Pakistan, the use of unprescribed antibiotics is a common practice, which results in the development of resistance to many of the pathogenic bacteria from antibiotics, including amoxicillin. Whereas, it was also reported that all of the probiotic isolates were sensitive to streptomycin, imipenem, and chloramphenicol.
Out of a total of 60 combinations of tests for bacteriocin production activity of six isolates against 10 different indicator bacteria, only two gave positive results on nutrient agar medium. This finding was comparable with some other reported probiotic strains, L. caseishirota, L. paracasei sub spp. tolerans, L. plantarum [64,65], and L. fermentum [66,67], which also did not produce bacteriocin against other bacteria. These results concluded that there is no existence of bacteriocin-like action [68], and inhibition of surrounding microbes was due to the acidic environment produced by probiotic LAB strains [69].
The present study showed that all isolates were able to tolerate salt concentrations up to 7%, but few were able to tolerate high concentrations. All isolates showed maximum growth with a 1% NaCl concentration, which is consistent with the findings of other studies [14,70].
In the present study, 0.1, 0.3, 0.5, and 1% bile salt concentrations were used for the growth of potential probiotic isolates. In a healthy human, 0.3% bile salt is present in the GIT, and for any bacteria to be used in probiotic production, it must tolerate this bile concentration [14,52,71,72]. All of the black olive isolates and a few of the green olive isolates gave maximum growth on 0.3% bile salt, and almost all of the isolates tolerate this bile concentration (up to 1%). This was because of a vital characteristic of Lactobacilli which enabled them to survive, grow, and exert their action in the GIT due to the action of bile salt hydrolase, thus reducing the toxic side effects of bile salts. Along with this, some components of food protect and promote resistance among strains to bile salts [68].  To achieve health benefits, probiotic foods must comprise an adequate amount of live bacteria, at least 10 6-7 CFU/g [73]. The ability to tolerate harsh conditions and viable cell counts of isolated potential probiotic bacteria were checked while incubating in artificial gastric juice with pH 2.2 for 30, 60, 90, 120 minutes, and 24 h. The experiments yielded ≤10 5 bacterial counts, and recent studies have provided significant data on the valuable immunological effects derived from dead probiotic cells [68]. The viability of isolates from black olives (Lactoccocus lactis MB418) and green olives (MB422) gradually decreased with incubation time, while black olive isolates MB416, and Lactobacillus crispatus MB417; and green olive isolates MB424, MB422 showed stability up to 24 h of incubation in acidic gastric juice. Results of the experiment predict that gastric juice in the GIT has fewer effects that could be hostile to most of the probiotic isolates [74]. Similar viability counts (3.4-5.6 logs CFU/ml) for the fermented olive isolates from the present study were also observed previously for L. plantarum, L. pentosus, and L. paraplantarum, showing 3-6 log CFU/ml viable bacterial cells [68].
In natural olives, organic acids can be added to create an optimal initial pH for the proliferation of LAB [69,75]. Olive samples used in the study also contained added lactic, ascorbic, and citric acids before packaging. The organic acid production ability of probiotic bacteria present inside the brine of black and green olives was determined to have an idea of the shelf life of the fermented food, which can be spoiled in terms of taste, texture, and smell due to altered lactic acid production. The Acidity of the brine of black and green olives remained the same throughout, but the brine of green olives was more acidic with a lower pH as compared to black olives. Because green olives were un-ripened, the bacteria employed for their fermentation had to work more to make them less bitter than for fully ripened black olives, for example, maturation of olives will condition phenolic compounds, sugar, and cell wall permeability [69].
In our study, the data analysis showed that one of the isolates, MB422, from green olives, is the least similar to the rest of the isolates. Isolates from black olives MB416 and green olives Carnobacterium divergens MB421 showed 84% similarity, which is 72% similar to Lactococcus lactis MB418. The cluster is 60% similar to other clusters, such as MB424 and Lactobacillus crispatus MB417 (75% similar).

Conclusion
It is concluded that isolated potential probiotic bacteria are well adapted to the olive surface or brine. Thus, the olives, either green or black, contained good microbial flora of probiotics and could be consumed as an effective probiotic food. As a result, the isolates from our study, specifically MB417, MB418, MB421, and MB424, demonstrated relatively good antipathogenic activity and survival in harsh conditions, suggesting that they could be considered viable "nextgeneration" probiotic candidates, which could be beneficial to the pharmaceutical industry.

Future Applications.
It would be advantageous to conduct a detailed study on the identification of isolates using molecular methods. The use of bacteriocin producers as starters is of considerable interest because bacteriocin production is a significant factor that helps to promote the safety and quality of fermented table olives and can also be used as natural antibiotics.

Data Availability
All the data is available in the manuscript.

Conflicts of Interest
The authors declare no competing interests.