Abstract: A total of 20 Lactobacillus strains isolated from fermented dates were tested for their probiotic potential by comparing their pH stability, resistance to low pH, and ability to tolerate bile salts. Out of 20 strains, 3 strains named Lactobacillus pentosus KAU001, Lactiplantibacillus pentosus KAU002, and Lactiplantibacillus plantarum KAU003 had a high tolerance of acids and bile salts and the capability to adhere to the intestinal wall. In addition, the three isolates were tested for their anti-oxidation, anti-glucosidase inhibition, cholesterol-lowering, and anti-inflammation properties. Among them, strain KAU001 and KAU002 inhibited α-glucosidase, lowered cholesterol level, inhibited nitric oxide production, and showed a higher anti-oxidative ability that was significantly better than strain KAU003. Both strains also significantly inhibited the release of inflammatory mediators such as TNF-α, IL-6, and IL-10 induced by LPS on RAW 264.7 macrophages (p < 0.001). The results indicated that KAU001 and KAU002 have the highest probiotic potential, potentially modulating metabolic health and reducing pro-inflammatory cytokines in response to allergic reactions.

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Keywords: Ajwa; Madinah; probiotics; inflammation; foods

1. Introduction 

Probiotics are dietary supplements that contain live microbial strains capable of colonizing the gastrointestinal tract (permanently or transiently) to improve human health [1,2]. Probiotics are beneficial when the native flora of the gastrointestinal tract is disrupted; exogenously supplemented probiotics might temporarily colonize the tract, and stabilize the microflora balance that ultimately restores the vital physiological function [1–5]. The term “probiotics” is generally limited to species belonging to the genus lactic acid bacteria (LAB), such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus sakei, and Lactobacillus plantarum, as important probiotics because of their strain-specific properties that are beneficial to health [6–15]. The current research in probiotics indicates that bacterial species should fulfill specific requirements, including resistance to high acid and bile concentrations, adhesion and establishment in intestinal epithelia, production of antimicrobial compounds, and modulation of immune responses [1,9,10,14,16,17]. 

Known for their ability to secrete various antimicrobial substances including bacteriocins, probiotics facilitate the immune response through the secretion of IgA to fight against potential pathogens, reduce allergic reactions, activate the mucosal barrier in the colon, increase the stability of commensal microflora, and decrease the adhesion of pathogens, as well as promote recovery [4,5,18–20]. Bacteriocins produced by L. gasseri are the best known of which gas sericin-A produced by L. gasseri LA39 is isolated from infant feces [21]. It is reported that Verdenelli et al., isolated L. rhamnosus IMC 501 from the fecal matter of an elderly Italian, exhibited high adhesion to the gastrointestinal tract, and discouraged the growth of pathogens, particularly Candida albicans [22]. Another probiotic isolated from koumiss, L. casei Zhang was also found to have high acid and bile salt resistance, gastrointestinal persistence, antimicrobial activity, as well as cholesterol-reducing properties [23]. Due to extensive commercial value and the health benefits contributed by probiotics, continuous isolation and characterization of new strains have become an emerging trend as these beneficial bacteria are currently the most potent multi-faceted biotherapeutics [1,9,10,14,18,24–31]. 

This study aimed to evaluate the probiotic potential of 20 Lactobacillus strains isolated from fermented dates, along with their resistance to high acid and bile concentrations. In addition, the selected isolates were investigated for their various functional properties,  such as adhesion to the intestinal wall, anti-oxidation, inhibition of glucosidase activity,  cholesterol-lowering, and anti-inflammation.

2. Materials and Methods 

2.1. Isolation and Identification of the Lactobacillus Strains

A total of 20 lactic acid bacteria strains were isolated from fermented Ajwa dates using BCP (bromocresol purple lactose) agar. The strains were further grown on MRS agar (de Man, Rogosa, and Sharpe), and pure culture strains were stored in 60% (v/v) glycerol at −80 ◦C [32]. Before each experiment, the cultures were activated by culturing twice in the MRS broth before use. Cell-free supernatant was prepared by culturing each LAB  isolate in MRS broth at 37 ◦C for 24 h, followed by centrifugation (10,000× g for 10 min)  and filter sterilization [7,8,18]. Strains sensitive to antibiotics were sent for 16S rRNA gene sequencing for identification [8].

2.2. Probiotic Properties of Selected Isolates in Gastrointestinal Tract Model

Survival gastric juice and bile salts: 

The resistance of selected LAB isolates to artificial gastric juice and bile salts was tested as per our previously described protocol [18]. Briefly, an inoculum with a cell concentration of 2.5 × 108 CFU/mL was prepared and inoculated in artificial gastric juice and bile salts. For gastric juice, the MRS was supplemented with pepsin (1000 units/mL) at a final pH of 3. Similarly, artificial bile was prepared by adding 5% pancreatin to MRS with 1% ox gall and the final pH was adjusted to 7.

2.3. Adhesion to HT-29 Intestinal Cells 

HT-29 cells were used to determine the ability of the selected LAB isolates to adhere to the intestine using the method described by Kim et al. [33]. The number of viable cells was determined by using the spread plate method on MRS agar [34].

2.4. In Vitro Study of the Functional Properties of the Lactobacillus Strains 

Determination of the antioxidant activity: 

The antioxidant activities of selected LAB isolates were determined by estimating the radical-scavenging ability using the 2,20 -casino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, the ferric-reducing antioxidant power (FRAP) assay, and the 2,2-diphenyl- 1-picrylhydrazyl (DPPH) assay. The ABTS, FRAP, and DPPH assays were performed by following the method described elsewhere [14,35–37].

2.5. nα-Glucosidase Inhibition 

Inhibition of α-GLU was performed according to the method previously described [14,32]  using α-GLU from Saccharomyces cerevisiae (Sigma, St. Louis, MO, USA) with a few modifications [32]. Briefly, 25 µL α-GLU (0.17 U/mL) and 50 µL of potassium phosphate buffer were mixed with 10 µL of the test sample. PBS and MRS were used as controls. Then, 5 mM of pNPG was added followed by incubation at 37 ◦C for 30 min. Finally, the enzymatic reaction was halted by adding 100 µL of 0.2 M Na2CO3. The absorbance was examined using a microplate at 405 nm. The percent inhibition of α-GLU for each strain was calculated by following Equation (1).

2.6. Determination of the Cholesterol-Reducing Activity 

Cholesterol-reducing activity was evaluated using the modified method described by Oh et al. [35]. The residual cholesterol was determined using the total cholesterol assay kit following the manufacturer's protocol. To serve as a negative control, the cholesterol in the uninoculated sterile broth was also analyzed.

2.7. Anti-Inflammatory Activity of LAB Isolates in RAW 264.7 Macrophage Cells

A murine macrophage cell line, RAW 264.7, was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in 10% FBS with 1% penicillin and streptomycin at 100 U/mL  and 100 µg/L, respectively, at 37 ◦C with 5% CO2 in a humidified atmosphere. The cells were sub-cultured and plated at 80–90% confluency. We treated RAW 264.7 cells (1 × 106  cells/mL)  with two concentrations of LAB (7 or 8 Log CFU/mL) in DMEM medium without antibiotics for 12 h before exposing the cells to 100 ng/mL LPS for 18 h to evaluate their anti-inflammatory activity. A medium-lacking-antibiotics was used for dissolving bacterial samples, and it was then added directly to the cell culture medium. Using the Griess reaction, NO was measured in cells induced by LPS. A quantitative real-time PCR was used to determine the mRNA expression of TNF-α, IL-6, and IL-10 released.

2.8. Statistical Analysis 

Based on independent experiments carried out in triplicate, all data are expressed as means + standard deviations (S.D.). A one-way analysis of variance (ANOVA) analyzed statistical differences between multiple groups using Duncan’s multiple-range test. The unpaired one-tailed Student’s t-test analyzed statistical differences between the two groups. p-values < 0.05 were considered statistically significant.

3. Results 

3.1. Screening and Isolation of Bacterial Strains from Fermented Ajwa Dates

Ajwa dates were acquired from a local market in Madinah, Saudi Arabia. The seeds were extracted and ground to powder. Then, the seed powder was mixed with the seedless dates and sterile water at a final concentration of 5% and then stored in glass jars. The containers were kept at room temperature for 48 h, followed by incubation at 10 ◦C for 10 days to complete the fermentation. A total of 20 strains of lactic acid bacteria (LAB)  were isolated from the fermented dates using BCP agar according to the method reported earlier [7,18]. After preliminary screening, the three selected LAB strains were identified as Lactobacillus pentosus KAU001, Lactiplantibacillus pentoses KAU002, and Lactiplantibacillus plantarum KAU003, and the sequences were submitted in GenBank with accession numbers ON514175, ON514180, and ON514177, respectively.

3.2. Probiotic Properties of the Lactobacillus Strains in the Gastrointestinal Tract Model 

Probiotics must be alive in the gastrointestinal tract to exert their beneficial effects. Therefore, as a supplement, they must first survive the passage through the high bile salts and acid-rich (low pH) zones in the gastrointestinal tract before colonizing it. In humans and most animals, the liver synthesizes bile acids from cholesterol, stores them in the gallbladder, and secretes them into the small intestine following a fatty meal. To qualify as probiotics, a novel lactic acid bacteria must be able to withstand high bile salts and low pH in the gut. Oxgall powder, a product derived from bovine bile, has been commonly used instead of human bile to measure potential probiotic bile tolerance abilities because of its similarity to human bile composition. All three isolates of Lactobacillus showed high tolerance to a hostile acidic pH of 3.0, with a survival rate of 92% and 97% in KAU002 and KAU003, respectively (Figure 1). Similarly, at a 1% concentration of bile salts, the isolates exhibited a survival rate ranging from 91% to 97.4% after incubation for 6 h.

The three isolates were tested to adhere to HT-29 human colon adenocarcinoma cells as a  prerequisite to colonization in the human intestine. Strains KAU001 and KAU002 exhibited higher adhesive abilities (exceeding 80%) in human colon cells. Properties such as higher tolerance to acidic pH, survivability in higher bile salt concentrations, and a higher adhesion rate to colon cells qualify the isolates as potential probiotic lactic acid bacterial strains.

3.3. Anti-Inflammatory Activities of the Lactobacillus Strains 

To determine the anti-inflammatory properties, a common antigen—lipopolysaccharides (LPS)—was used to stimulate RAW 264.7 macrophage cells, and these cells were inoculated with Lactobacillus isolates. MTT assays showed no cytotoxic effect of lactobacillus strains on RAW 264.7 cells (data not shown). The bacterial isolates (KAU001 and KAU002) at two concentrations—107 and 108 CFU/mL—significantly inhibited the production of nitric oxide (NO) compared to the control LPS-stimulated cells (Figure 2). In addition, the amount of pro-inflammatory cytokine expression of TNF-α, IL-6, and IL-10 in LPS-stimulated RAW 264.7 was quantified using qRT-PCR. Lactobacillus strains KAU001, KAU002, and KAU003  significantly reduced the expression of pro-inflammatory cytokines in a concentration-dependent manner (Figure 3).

4. Discussion

Probiotics, mainly comprised of lactic acid bacteria, are known to provide specific health benefits to the host through the colonization of the gastrointestinal tract [38]. To exert such biotherapeutic effects, probiotic strains should survive the passage through the gastrointestinal tract and colonize the small intestine and colon for a sufficient period [18,38]. Strains such as KAU001 and KAU003 were the most acid and bile tolerant, and their adhesion to HT-29 cells was significantly higher.

Some LAB isolates from chicken caecum show a limited survival of 60% to 80% after being exposed to an acid environment with pH 2–2.5 for 3 h [2,4,39–41]. In contrast, a survival rate as high as 92.75~97.26% was observed in this study. The natural human defense mechanism against pathogens in the intestinal tract is through the production of hydrochloric acid which lowers the stomach pH. Therefore, strains with acid tolerance are believed to survive the transit through the upper gastrointestinal tract. Whereas in the small intestine and colon, a high abundance of bile salt is also a protective barrier to prevent attachment of pathogenic bacteria, hence survival in bile is considered a crucial factor for probiotics to colonize the gut [17]. The mechanism of probiotics in counteracting the damaging effect of bile salt is through the production of the enzyme bile salt hydrolase (BSH),  which can break down conjugated bile salts, thereby reducing toxicity [23,40]. Another important requirement for probiotic strains is the ability to adhere to intestinal epithelial cells, which enables them to colonize the gut, compete with pathogens, and confer benefits to the host [42,43]. Among all the strains tested, KAU001, KAU002, and KAU003 depicted a high ability to survive the gastrointestinal tract and adherence to colon cells, conferring them as potential probiotic candidates.

KAU001 and KAU002 were found to have significant antioxidant potential, particularly KAU001. These results underpin that both LAB strains are highly effective in scavenging peroxyl radicals. Probiotics with high antioxidant potential may help to reduce the oxidative damage caused by free radicals, thereby promoting health benefits such as slowing down cell aging as the aging process is highly associated with accumulated oxidative stress. Additionally, KAU001 and KAU002 inhibited α-glucosidase activity, which is  one of the important determinant factors in metabolic diseases such as diabetes and obesity

Reduction of α-glucosidase could indicate the ability of these strains in reducing intestinal sugar absorption, therefore potentially reducing the risk of diabetes. Besides,  both strains also significantly lowered cholesterol levels, which may help to reduce the risk of high cholesterol levels and cardiovascular disease. It was postulated that BSH produced by LAB strains is responsible for the anti-cholesterol properties through the deconjugation of bile salts. Deconjugated bile salts are less reabsorbed by the intestines, and they are excreted through feces thus the replacement of new bile salts from cholesterol will lower the serum cholesterol level [30].

In response to LPS stimulation in RAW 264.7 macrophages, the cells produced NO as a toxic defense molecule to fight off pathogens, and this led to inflammation. Treatment with Lactobacillus strains reduced the NO production in antigen-stimulated macrophages,  in parallel with increasing Lactobacillus concentrations. Coherently, the expression levels of TNF-α, IL-6, and IL-10 also decreased with the increasing concentration of KAU001 and KAU002, justifying the anti-inflammatory properties of these strains. In an allergic reaction stimulated by an antigen such as allergic rhinitis and atopic dermatitis, the severity of the disease can increase over time with repeated exposure, and it can cause uncontrolled inflammation [41]. Typically, anti-inflammatory drugs such as steroids are used to control these diseases; however, the drugs are accompanied by many side effects. Probiotics are one of the safer alternatives to counteract these hypersensitivity problems as the beneficial bacteria have excellent anti-inflammatory properties.

5. Conclusions

In summary, the Lactobacillus strains isolated from the fermented dates showed that all three strains, KAU001, KAU002, and KAU003 could survive the GI tract and adhere to the intestinal mucosa. Strains KAU001 and KAU002 were potential probiotics with an important health-promoting effect in the aspect of metabolic health and hypersensitivity reactions due to their ability to exert an anti-oxidation effect, an inhibition of α-glucosidase activity, and a cholesterol-reducing and an anti-inflammatory effect. These plant-based  strains should be considered for further characterization and commercialization in the food  and dairy industry

Author Contributions: Conceptualization, I.A.R.; methodology, A.A.M., A.F. and H.M.A.; software, S.-H.Y. and I.A.R.; validation, Y.-H.P., I.A.R. and Y.-Y.H.; formal analysis, A.A.M.; investigation, A.A.M.;  resources, A.F., H.M.A., S.-H.Y., Y.-H.P. and I.A.R.; data curation, A.A.M.; writing—original draft preparation, A.A.M., I.A.R. and Y.-Y.H.; writing—review and editing, Y.-H.P., Y.-Y.H. and I.A.R.; visualization, Y.-H.P.; supervision, I.A.R.; project administration, A.A.M. and I.A.R.; funding acquisition, A.A.M. and H.M.A. All authors have read and agreed to the published version of the manuscript.

Funding: This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. (DF-552-130-1441). The authors, therefore, gratefully acknowledge DSR’s technical and financial support.

Institutional Review Board Statement: Not applicable. 

Informed Consent Statement: Not applicable. 

Data Availability Statement: The data generated is cited in the manuscript. 

Conflicts of Interest: The authors declare no conflict of interest.

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