Probiotics, recognized for their nutraceutical benefits, require verification of their safety for human health before they can be endorsed as beneficial gut bacteria. The market is replete with various probiotic strains, presenting an opportunity to utilize this readily available resource for isolating and crafting new, improved probiotic strains with significant medical benefits. This approach is a promising strategy for the development of superior probiotic strains [43]. The genus Lactobacillus represents a broad and varied collection of gram-positive, non-sporulating, facultatively anaerobic bacteria. It includes species such as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus casei, and Lactobacillus reuteri, among others. This sort assumes an imperative part in food maturation and can likewise be viewed as in the gastrointestinal (G arrangement of people and creatures in factor sums [44]. Eight examples of milk were randomly collected from the markets in Egypt. The initial samples were anaerobically plated onto MRS agar after being serially diluted. A total of 16 lactic acid bacteria isolates were retrieved from the previously mentioned samples and subjected to rapid preliminary identification. The total discoveries of morphological and biochemical tests. The best organism has been selected based on its ability to grow at a 2% salt concentration and yield the highest absorbance. The morphological and biochemical tests of the best organism are introduced in Table 2. Staining revealed the gram-positive nature of the isolate, which had a purple or violet color. The isolate was rod-shaped and had long, rounded ends. They showed up generally as a chain of 3–4 cells, either single or two by two (Fig. 1). The hanging drop strategy showed that the microscopic organisms were non-motile, which is one of the interesting attributes of Lactobacilli. This may be because of the shortfall of extraordinary propeller-like flagella in Lactobacilli answerable for motility. Similar findings were made by Forouhandeh [45] in the isolation of Lactobacillus species from various dairy products. The absence of air bubbles indicated that the isolated microbes were catalase-negative, thereby incapable of breaking down hydrogen peroxide to release oxygen. It is well established that Lactobacillus species do not produce catalase. Comparable findings were documented by Mithun et al. [46].
The preliminary optimization, including pH, temperature, incubation period, carbon, nitrogen sources, NaCl, ethanol, and bile salt, was used in this study. The L. plantarum strain was incubated at varying temperatures, from 20 ºC to 45 ºC. The findings indicated that the optimal growth temperature for the L. plantarum isolate was 30°C, with an enzyme activity level of 1.879 ± 0.130. Conversely, the lowest enzyme activity, 0.513 ± 0.01, was observed at a lower temperature of 20 oC, with enzyme activity diminishing sharply as temperatures approached this lower limit. Consequently, the study identified that enzyme activity decreased at temperatures below 30°C, while increasing up to 35 oC, establishing 30 oC as the ideal temperature for cultivating the L. plantarum strain (Fig. 2 (A)). The best incubation duration for producing the L. plantarum strain was detected at 24 hours, with the most conducive conditions for its growth under static conditions being 1.796 ± 0.012 (Fig. 2 (B)). The study also explored the effect of pH levels on the production of the L. plantarum strain, revealing that the most favorable pH for its production was 6, with an optimal yield of 2.123 ± 0.27 (80%) (Fig. 2 (C&D). Furthermore, the research investigated the impact of different carbon and nitrogen sources on the strain's production. It was found that glucose combined with yeast extract resulted in the highest production levels, with values of 2.971 ± 0.020 and 2.414 ± 0.26, respectively (Fig. 2 (E and F)). The optimal production of the L. plantarum strain was achieved using lactose as the carbon source and ammonium nitrate as the nitrogen source. Additionally, the study assessed the tolerance of the L. plantarum strain to NaCl, ethanol, and bile salts, recording values of 2.987 ± 0.2 (93%) at 2% (Fig. 2 (G&H)), 1.877 ± 0.2 (70%) at 2.5% (Fig. 2 (I&J)), and 2.325 ± 0.2 (75%) at 0.1% (Fig. 2 (K&L)), respectively, demonstrating the strain's resilience to these conditions. Similarly, Coulon et al. [47] have reported that Lactobacillus casei ATCC393 found 35 oC to be the optimal temperature for the production of β-glucosidase among the various temperature ranges. Also, the ideal temperature for compound creation from yeast, such as Debaryomyces pseudopolymorphus [48] and Saccharomyces cescerevisiae [49], was 40°C, respectively. The ideal pH for Lactobacillus rhamnosus CRL 98 was 6.4, which was like the L. plantarum strain, which expressed that various upsides of ideal pH were accounted for in various types of lactic corrosive microscopic organisms. Lactobacillus mesenteroides [50] and Lactobacillus plantarum [51] grow at an optimum pH of 5.0 and 5.4, respectively. The metabolism of carbon sources releases energy, which is utilized by the organism for its growth and development. The influence of different carbon sources on the production of L. plantarum strains was investigated in this study. It was discovered that lactose serves as an efficient carbon source for cultivating the L. plantarum strain. Interestingly, the organism demonstrated significant growth on xylose, resulting in the highest protein activity in the culture broth compared to other carbon sources tested. This observation indicates that the Lactobacillus plantarum strain is capable of thriving or adapting to diverse environmental conditions. In certain examinations on glycosidase chemicals from different sources, Grimaldi et al. [52] have announced that the presence of glucose or fructose diminishes the exercises. Also. when culture in a fluid medium contained corn grain, the greatest aggregate β-glucosidase creation was accounted for, cellulose and glucose prompted elevated degrees of β-glucosidase creation, Pediococci sp. creation of β-D-glucopyranosidase movement was unequivocally worked on by both glucose and fructose, and furthermore, it was presented as an expected supportive source from which higher compounds could be cleansed [53]. L. plantarum strain was developed on carbon wellsprings of focus 1% m/v.
In many microorganisms, both organic and inorganic nitrogen forms are metabolized to produce amino acids, nucleic acids, proteins, and components of the cell wall [54]. It was found that the use of ammonium nitrate resulted in the highest production levels of the Lactobacillus plantarum strain among various organic and inorganic nitrogen sources tested, with urea-containing medium showing the lowest activity. Regarding the impact of different ethanol concentrations on the optical density and viability of the L. plantarum strain, it was observed that a 2.5% (v/v) ethanol concentration yielded the highest activity compared to other tested concentrations. Furthermore, Spano et al. [55] reported that the β-glucosidase gene from Lactobacillus plantarum was inhibited by 12% (v/v) ethanol [52]. Notably, ethanol was found to activate the enzyme in wine at concentrations above 15% (v/v). In contrast, Grimaldi et al. [52] noted that the stimulatory effect of 4% v/v ethanol is more pronounced at lower concentrations, aligning with previous findings for Oenococcus oeni and other yeast biomasses. The study led with various groupings of NaCl for the creation of L. plantarum showed the greatest worth at convergence of 2% NaCl. It has been demonstrated that sodium ions in the surrounding environment are necessary for efficient membrane transport. The utilization of NaCl in a concentrate by Damaso et al. [56] demonstrated that it is necessary for the synthesis of another enzyme, xylanase.
Lactobacilli was identified genetically through PCR amplification and sequencing of 16S rDNA using two bacterial universal primers, as mentioned. The PCR amplicon was purified from gel, then sequenced and aligned through NCBI BLASTn for firmly related sequences on the NCBI gene bank database; this indicates that it belonged to Lactobacillus plantrum, and its phylogenetic tree was designed through MEGA 11 with the most related sequences as described in Fig. 3. This technique was used by Khedr et al. [41] to identify Lactobacillus delbrueckii. Simmalry, Abdel Ghany et al. [42] used this technique to identify Lactobacillus acidophilus.
Table 2
The morphological and biochemical characterization of the best organism.
No. | Characteristics | Results |
Morphological tests |
1 | Gram staining | +ve |
2 | Shape | Rod |
3 | Colony morphology | Circular, white, glistering, convex. |
4 | Motility | Non motile |
Biochemical tests |
1 | Catalase | - |
2 | Citrate test | - |
3 | NH3 from arginine | - |
4 | Hydrogen sulphide production | - |
5 | Indole production | - |
6 | Methyl red reaction | + |
7 | Oxidase test | - |
8 | Urease test | - |
9 | Voges Proskauer Reaction | - |
10 | Glucose utlization | + |
11 | Glucose (Gas) (Co2) | - |
12 | Sucrose utlization | + |
13 | Lactose utlization | + |
14 | Maltose utlization | + |
15 | Mannitol utlization | + |
16 | Arabinose utlization | - |
17 | Salicin utlization | + |
+, positive result; -, negative result.
from the NCB Gene Bank site.
High-Performance Liquid Chromatography (HPLC) is a critical and versatile analytical method extensively employed across various fields. It is utilized for both the separation and quantification of organic and inorganic substances in a wide array of samples, including those from industrial, pharmaceutical, food, and environmental sources. Moreover, it is increasingly applied to biological samples and the extraction of natural products. Through HPLC, compounds are differentiated based on their interactions with the mobile phase solvent and the solid material within a tightly packed column, under high pressure. This technique is gaining recognition as a preferred method for fingerprinting analysis, crucial for the quality assurance of herbal products [57]. Various researchers and scholars have highlighted the application of HPLC in the characterization and quantification of secondary metabolites in plant extracts, specifically targeting phenolic compounds, steroids, flavonoids, and alkaloids [58–60].
Flavonoids constitute a vast group of polyphenolic compounds, all structurally derived from the base molecule flavone and produced by plants. These substances, found in fruits and vegetables, are recognized for their broad and significant health benefits, which include radical scavenging and metal chelating activities. The antioxidant properties of flavonoids in vitro stem from their ability to mitigate free radical formation, leading to various biological effects. Numerous studies have highlighted that flavonoids such as rutin, kaempferol, quercetin and apigenin, among others, are renowned for their anti-inflammatory, anti-allergic, antithrombotic, hepatoprotective, antispasmodic, and anticancer properties [61, 62]. The biological activities, such as their antimutagenicity, antibacterial, antiviral, anti-inflammatory, and apoptotic properties, among others, must be justified by distinguishing and quantitating such mixtures [63]. Syringic acid is accepted to have various advantageous natural exercises, including the insurance of the mind, heart, and liver, as well as anticancer, antimicrobial, against aggravation, antidiabetic, hostile to nitrosative, and cell reinforcement properties [64]. Coumaric acid demonstrates a wide range of bioactive properties, including antioxidant, anti-inflammatory, anti-mutagenic, anti-ulcer, antiplatelet, and anti-cancer activities. Besides, it plays a role in mitigating atherosclerosis, oxidative damage to the heart, damage to ocular tissues caused by UV light, neuronal injury, anxiety, gout, and diabetes [65]. According to the current study, CA may alleviate diabetes by increasing its immunomodulatory effect and defending against oxidative stress and inflammation. Quercetin has drawn expanding consideration because of its cancer prevention agent, antibacterial, and anti-inflammatory effects [66, 67].
The results from the study on L. plantarum indicate that the strain tested is capable of producing secondary metabolites with promising antimicrobial, antioxidant, and anti-inflammatory properties (Table 3 and Fig. 4). However, to validate L. plantarum's potential as a biomedical agent, in vivo studies will be necessary.
Table 3
Bioactive compounds isolated from L. plantarum.
Peak No. | RT | Type | Area % | Compound name | Molecular weight (g/mol) | Molecular formula |
1 | 3.592 | BV | 74.5124 | Gallic acid | 170.12 | C7H6O5 |
2 | 4.308 | BB | 10.7387 | Chlorogenic acid | 354.31 | C16H18O9 |
3 | 4.494 | - | 0.0000 | Catechin | 290.26 | C15H14O6 |
4 | 5.622 | BV | 5.2960 | Methyl gallate | 184.147 | C8H8O5 |
5 | 5.904 | VV | 2.0099 | Coffeic acid | 180.16 | C9H8O4 |
6 | 6.161 | VB | 3.1779 | Syringic acid | 198.17 | C9H10O5 |
7 | 6.649 | - | 0.0000 | Pyro catechol | 110.1 | C6H6O2 |
8 | 6.925 | - | 0.0000 | Rutin | 610.517 | C27H30O16 |
9 | 7.411 | BB | 0.4946 | Ellagic acid | 302.197 | C14H6O8 |
10 | 8.702 | - | 0.0000 | Coumaric acid | 164.16 | C9H8O3 |
11 | 9.123 | - | 0.0000 | Vanillin | 152.15 | C8H8O3 |
12 | 9.756 | - | 0.0000 | Ferulic acid | 194.18 | C10H10O4 |
13 | 10.263 | BB | 5.4176 | Naringenin | 272.257 | C15H12O5 |
14 | 11.846 | - | 0.0000 | Rosmarinic acid | 360.318 | C18H16O8 |
15 | 16.021 | - | 0.0000 | Daidzein | 254.23 | C15H10O4 |
16 | 17.331 | - | 0.0000 | Querectin | 302.236 | C15H10O7 |
17 | 19.263 | - | 0.0000 | Cinnamic acid | 148.1586 | C9H8O2 |
18 | 20.610 | - | 0.0000 | Kaempferol | 286.23 | C15H10O6 |
19 | 21.205 | - | 0.0000 | Hesperetin | 302.27 | C16H14O6 |
Anti-Neoplastic activity of Lactobacillus plantarum
Figure 5 illustrates changes in cell morphology and shape within a monolayer culture as an initial and distinct effect observed following exposure to L. plantarum, captured using an inverted light microscope. The inhibitory effect of L. plantarum on human colon cancer cells (HCT116), along with the degree of cell suppression, was confirmed using the MTT assay at different concentrations ranging from 1000 to 31.25 µg/mL. Significantly, the IC50 value, indicating the concentration needed to inhibit 50% of the cancer cells (HCT116), was found to be 100.11 µg/mL. Lactobacillus strains are commonly recognized for their health-promoting roles as microbial food supplements, with benefits such as enhancing gut health, boosting the immune system, and lowering the risk of certain cancers [68]. Regular consumption of yogurt and other probiotic dairy products has been suggested to inhibit the growth of colon cancer cells [69]. The surface components of Lactobacillus strains have shown anticancer activities. This study explores the anti-cancer capabilities of L. plantarum. Previous research has indicated the role of autophagy in cancer prevention and treatment. This investigation found distinctive morphological and biochemical markers of autophagy, such as autophagic vacuoles and acidic vesicular organelles, in HCT116 colon cancer cells treated with L. plantarum, suggesting that L. plantarum triggers autophagic cell death in HCT116 colon cancer cells [70].
DNA fragmentation ability of lactobacillus plantrum
Our research demonstrates that L. plantarum significantly contributes to triggering cell death in cancer cells via DNA fragmentation, indicative of necrosis. This observation is supported by gel electrophoresis findings, which displayed DNA fragmentation in cells treated with L. plantarum, in stark contrast to the intact DNA in untreated (control) cancer cells, as illustrated in Fig. 6. This finding differs from the behavior of nuclear DNA in cancer cells. Moreover, Choi et al. [71] discovered that soluble polysaccharides from the cell wall of L. acidophilus 606 inflicted damage on HT-29 cancer cells, a phenomenon largely attributed to the initiation of apoptosis rather than necrosis, as evidenced by nuclear DNA fragmentation and the lack of PI staining. This marks a pioneering instance of cancer cell apoptosis triggered by Lactobacilli-derived polysaccharides.
Additionally, proteomic analysis revealed that polysaccharides from L. acidophilus 606 significantly affected the expression of proteins such as the Bcl-2-interacting mediator and cell division cycle proteins. These findings underline the potent antioxidative and anticancer properties of soluble polysaccharides from L. acidophilus 606 against various cancer cell lines. The potential of these polysaccharide components to be integrated into foods or used as supplements in cancer therapy is significant [72, 73]. Furthermore, L. plantarum is shown to modulate the expression of crucial genes like AKT, PTEN, BAX, and TLR4, which are involved in apoptosis and anti-apoptosis mechanisms in the AGS gastric cancer cell line [74].
Anti-inflammatory activity of Lactobacillus plantarum
The evaluation of L. plantarum's anti-inflammatory effects was conducted through its capacity to suppress hypotonicity-induced hemolysis and perform hemolytic assays in vitro. The results showed that L. plantarum significantly reduced hemolysis by 97.7% at a concentration of 1000 µg/mL, nearly matching the 99.5% effectiveness of indomethacin, a widely recognized anti-inflammatory medication, at the same concentration (Fig. 7). On the contrary, the hemolytic activity of L. plantarum peaked at 14.3% at a concentration of 100 µg/mL and then diminished to 1.4% at 1000 µg/mL (Fig. 8). These findings underscore the potential of L. plantarum as an effective anti-inflammatory agent [75]. Similarly, L. casei and L. acidophilus have been observed to significantly alleviate paw swelling in rats, indicating their anti-inflammatory properties. Research conducted by Ganji-Arjenaki and Rafieian-Kopaei [76] has shown the efficacy of various Lactobacillus strains in the treatment of inflammatory bowel disease. In contrast, a study found that exopolysaccharides from Bacillus circulans exhibited a 92% anti-inflammatory effect, while EPS from Pseudomonas mendocina AB1 showed a lesser effect of 59.07% [77]. These comparative insights call for further investigation to elucidate the mechanisms of EPS in protein protection and their application in developing new anti-inflammatory treatments [78].
Figure 7. Effect of L. plantarum on HRBC hemolysis and membrane stabilization
The L. plantarum strain we studied exhibited notable anti-inflammatory properties by reducing the expression of two critical markers of inflammation in human cells: CRP and TLR2. Specifically, the expression levels of CRP and TLR2 decreased by one and eight times, respectively, in the cell line treated with our strain, which was fermented for 72 hours and then incubated for five hours at 37°C. These findings are consistent with in vitro research conducted by Borchers et al. [79], which indicates that Lactobacillus plantarum 299v may reduce inflammation in humans through the suppression of TLR activation. Additionally, the interaction observed between human peripheral blood mononuclear cells (PBMCs) and L. plantarum species underscores L. plantarum's potential to modulate PBMC responses [80].
Antioxidant activity of Lactobacillus plantarum
Lactic acid bacteria (LAB), including those with antioxidant enzymes, are vital for enzymatic defense against oxidative stress. The antioxidant potential of L. plantarum was evaluated over a spectrum of concentrations from 1000 to 1.95 µg/mL, as shown in Fig. 9. The results indicated that L. plantarum exhibited notable antioxidant efficiency, with activities of 71.8% and 93.8% at concentrations ranging from 125 to 1000 µg/mL, respectively. However, at lower concentrations of 7.81, 3.9, and 1.95 µg/mL, the observed antioxidant activities were 45.1%, 34.2%, and 27.2%, respectively, when compared to ascorbic acid, the standard reference used. Moreover, L. plantarum has demonstrated its capacity to counteract free radicals. Our study highlights that L. plantarum strains AR113, AR269, AR300, AR501, and P. pentosaceus AR243 showed considerable resilience against hydrogen peroxide [81]. In this context, L. plantarum was recognized for its profound antioxidant activity. This is in line with the findings of Li et al. [82], who found that L. plantarum strains from traditional Chinese fermented foods possess antioxidant capabilities, with L. plantarum C88 showcasing optimal hydroxyl radical and DPPH scavenging activities against hydrogen peroxide at a density of 1010 CFU/ml. The DPPH scavenging efficiency of our isolates surpassed those documented by Benattouche et al. [83], who reported antioxidant activities ranging from 16–56% for exopolysaccharides derived from various yogurt LABs at a concentration of 1000 µg/mL.
Gene expression was induced by Lactobacillus plantarum.
Moreover, L. plantarum has been found to modulate the expression of the antioxidant markers SOD1 and SOD2, enhancing their levels by 65% and 74.2%, respectively. It also significantly boosts the gene expression of TLR2 by 133% compared to the control, while reducing CRP expression by 33.3%, as depicted in Figs. 10A and B. These results are in line with findings by Rolfe [84], who observed that LAB supplementation could mitigate oxidative stress in piglets. Many probiotics, particularly LAB, are increasingly recognized as alternatives to antibiotics and as therapeutic options for managing post-weaning syndrome. They achieve their beneficial effects through various actions, including immune system activation, pathogen invasion blockade, and antimicrobial substance production. Supplementation with LAB notably enhances (p < 0.05) the expression of Btk, HO-1, Nrf2, TLR4, and TLR2 in the jejunum, in contrast to the LPS-only group. Protein expression of TLR4, Btk, and Nrf2 in the ileum of LPS-challenged piglets was also elevated (p < 0.05) following LAB supplementation [85]. Additionally, LAB helps shield the intestine from oxidative damage in animals by activating antioxidant enzymes and preserving redox homeostasis [13].
Four interest gene sequences were aligned against the most related sequences in the NCBI database; based on their sequences, phylogenetic trees were constructed as shown in Fig. 11.