1 Introduction

Phytic acid (PA), also known as myo-inositol hexakis dihydrogen phosphate or phytate, is primarily stored as salts in plant seeds, cereal grains, nuts, legumes, and oil seeds. These salts serve as the main storage form for both phosphate and myo-inositol, constituting approximately 1–5% of the seed weight [1]. Among these sources, cereals contain the highest concentration of phytic acid. Phytic acid forms complexes with divalent cations including Ca2+, Mg2+, Fe2+, Zn2+, Mn2+, and amino group derivatives found in protein molecules. This complexation reduces the solubility and absorption of these nutrients from the diet, thereby decreasing their bioavailability [2]. This property of phytate leads to adverse effects on nutrition and deficits in micronutrients [3]. Phytate can be removed by non-enzymatic as well as enzymatic approaches [4]. Enzymatic methods most effectively reduce phytate content, particularly when fermentation is carried out using a combination of bacterial cultures at a temperature of 30 °C for a duration of 72 h [5]. Phytases are the enzymes responsible for the hydrolysis of phytate. Phytase (myo-inositol hexakis phosphate phosphohydrolase, EC 3.1.3.8/EC 3.1.3.26) is an enzyme that hydrolyzes phytate in a stepwise process, releasing soluble inorganic phosphate, lower forms of inositol phosphates, and myo-inositol [6,7,8]. Phytases are found in many different living organisms, such as plants, animals, and microbes. However, humans do not naturally produce phytases, and their small intestines do not have a strong population of microorganisms capable of producing phytases [9]. Phytases generated from microorganisms have significant promise for application in the food industry [10]. Nevertheless, there is a subject of debate regarding the safety of utilizing microorganisms or their enzymes as dietary supplements for humans [11]. Therefore, ongoing attempts are being made to identify and separate microorganisms that produce phytase and are suitable for human consumption [12]. On the other hand, high cost and low efficient production of phytases from plants are considered major problems [13]. Furthermore, the higher specificity, pH, and thermal stability of microbial phytases compared to plant phytases have made the microbial phytases more widely studied for industrial purposes [14].

Lactic acid bacteria (LAB) are a significant group of Gram-positive bacteria that are involved in many industries and applications due to their potency and generosity in producing metabolites as well as their generally recognized as safe (GRAS) status [15]. Moreover, LAB are known for their probiotic characteristics and other biological activities such as antimicrobial activity, maintaining serum cholesterol levels, alleviating lactose intolerance. They also possess anticancer activity [16, 17]. Many metabolites are produced by LAB, including antimicrobial peptides (bacteriocins), exopolysaccharides, organic acids, ethanol, diacetyl, hydrogen peroxide, CO2, reuterin, acetaldehyde, acetoin, and enzymes such as phytases [18].

Therefore, phytases derived from LAB can be utilized to release metal ions by breaking down phytic acid in phytate-rich food. Over the past twenty years, there have been studies on LAB that produce phytase. These LAB have been analyzed and found to have potential uses in many food applications. L. casei DSM 20011 and L. plantarum W42 were reported to produce phytase. Pediococcus pentosaceus KTU05-8 and KTU05-9 strains were also reported to produce extracellular phytase [19,20,21]. The majority of LAB are recognized for their ability to create intracellular phytases. Recently, many findings have indicated that LAB can produce extracellular phytases [22, 23]. Considering the multitude uses of phytic acid-rich food and its potential health benefits, phytic acid-rich food by-products, such as rice and wheat bran, are used as an economical alternative in human, animal, and fish nutrition due to the need for a circular economy based on sustainable food production and waste recovery [24]. Therefore, the aim of this study was to screen for a potent phytase-producing LAB, investigate its safety and probiotic characteristics, and evaluate the reusability of its immobilized active cells for continuous phytase production.

2 Materials and methods

2.1 Isolation and presumptive identification of LAB

Samples used to isolate LAB were collected from different Egyptian food and non-food sources, with soil samples collected manually and food samples purchased from the market. All samples were collected in sterile plastic containers and transferred to a cool box (4 °C) until being examined. For bacteria isolation, one milliliter (or one gram) of each sample was suspended in sterile saline solution (0.85% NaCl), and ten-fold dilutions were prepared [25]. Appropriate dilutions were inoculated into de Man, Rogosa, and Sharpe medium (MRS), then plates were incubated at 37 °C for 24–48 h in anaerobic jars under anaerobic conditions. Bacterial colonies were re-plated and purified on MRS agar plates until pure colonies were obtained. The resulting isolates were stored at − 80 °C in MRS with 50% (v/v) glycerol as a frozen stock, then propagated in MRS broth medium at 37 °C for 24 h before use. LAB isolates were initially identified based on their colony morphology, catalase reaction, Gram staining, and spore formation [26]. Colonies showing Gram-positive and catalase-negative reactions, and being non-spore-forming and non-motile, were presumptively identified as LAB and were selected for further studies. For the catalase reaction, drops of a 3% hydrogen peroxide solution were placed on 24 h-old vegetative cells of each isolate. The appearance of bubbles means the presence of catalase in the cells (catalase positive) [26].

2.2 Screening for phytase-producing LAB and quantitative determination of phytase activity

Isolates presumptively identified as LAB were cultured in a modified MRS broth with the addition of 0.725 g/L phytic acid dipotassium salt (C6H16O24P6K2, Sigma-Aldrich, USA) at 37 °C for 72 h using a 10% inoculum [27]. After this incubation period, the extracellular phytase activity was assessed in the culture supernatants.

To determine phytase activity, the amount of inorganic phosphate released from sodium phytate was measured following a method described by Haros et al. [28]. The assay involved a mixture of 400 μL of 0.1 M sodium acetate-acetic acid buffer (pH 5.5) containing 1.2 mM sodium phytate and 200 μL enzyme. After incubation at 50 °C for 30 min, the reaction was halted by adding 100 μL of 20% trichloroacetic acid solution, and the liberated inorganic phosphate was analyzed using the ammonium molybdate method [29] at an absorbance of 405 nm. Phytase activity was quantified as the enzyme's capacity to produce 1 μM of inorganic phosphorus per minute at 50 °C [30].

2.3 Probiotic features of the selected isolates

2.3.1 Stress tolerance of LAB isolates

The selected isolates were assessed for their probiotic characteristics and stress tolerance in the laboratory. Stress tolerance tests were conducted following the methodology outlined by Parente et al. [31]. To start, overnight cultures of the isolates cultivated in MRS broth at 37 °C were collected by centrifugation, washed twice with sterile 0.2 M sodium phosphate buffer (pH 7.0), and then re-suspended in the same buffer to achieve a standardized optical density (OD) of 1.0 at 600 nm. Various stress conditions were tested, including acidic stress (pH 2.5 in MRS medium and pH 3.5 in glycine–HCl buffer), alkaline stress (pH 9.0 in glycine NaOH buffer), osmotic stress (3 M NaCl), osmotic stress in 3 M NaCl, oxidative stress in H2O2 (0.05% v/v), and heat stress at different temperatures (55 and 70 °C for 30 and 15 min, respectively) in 0.2 M sodium phosphate buffer, pH 7. Bacterial cells were exposed to each stress condition for a specific duration (3 and 6 h) at room temperature, followed by sub-culturing in MRS broth and subsequent incubation at 37 °C for 24 h. Furthermore, detergent stress was tested by inoculating LAB isolates in MRS broth supplemented with various substances such as Tween 80 (at 0.2%), bile salts (at 0.05 and 0.1%), and pancreatic enzymes (pancreatin from porcine pancreas, Sigma) (at 1.5 g/L) and incubating the cultures at 37 °C for 24 h. Isolates with low tolerance to bile salts were further cultured in increasing concentrations of bile salts until obtaining variants capable of growing in 0.1% bile salts for 24 h. Control experiments involved suspending bacterial cells in sodium phosphate buffer and storing them at 4 °C for an hour, considering them 100% viable. All experiments were carried out in duplicate to ensure consistency and accuracy of the results.

2.3.2 Cell surface hydrophobicity of LAB isolates

The hydrophobicity of bacterial cells, indicating their tendency to adhere to hydrocarbons, was assessed following the method detailed by Vinderola and Reinheimer [32]. In brief, 24 h-old cultures of the isolates were collected through centrifugation at 5000 rpm for 10 min at 4 °C, washed twice with 0.1 M sodium phosphate buffer at pH 7.0, and then re-suspended in the same buffer. The cell suspension was standardized to an optical density (OD) of around 1.0 at 600 nm. Subsequently, three milliliters of these bacterial suspensions were mixed with 0.6 ml of the non-polar solvent, n-hexadecane (from Merck, Germany), and agitated for 2 min. The mixture was left for an hour at 37 °C to allow separation into layers. The OD at 600 nm of the resulting aqueous phase was recorded. The decrease in absorbance in the aqueous phase was used to calculate the cell surface hydrophobicity (H%) using the formula:

$$\text{H }\%= \frac{{OD}_{0}-OD}{{OD}_{0}}* 100$$

where OD₀ and OD represent the optical densities before and after extraction with n-hexadecane, respectively. This experiment was duplicated, and the results are presented as mean values with standard deviation.

2.3.3 Antioxidant activity of LAB isolates

The method for assessing DPPH (1-diphenyl-2-picrylhydrazyl) scavenging activities of the chosen isolates was implemented according to the protocol outlined by Lee et al. [33] with minor modification. The process involved combining equal volumes of ethanolic DPPH solution and bacterial cell suspension, which had been cultured for 24 h and adjusted to a specific optical density (standardized to obtain a final OD600 of 1.0). A control was prepared using un-inoculated MRS instead of the bacterial suspension. 0.01 g ascorbic acid was used as a positive control. After vigorous mixing, the samples were kept in dark conditions at 37 °C for an hour. Subsequently, the absorbance of each mixture was determined using spectrophotometry at a wavelength of 517 nm. The scavenging activity was calculated as:

$$\% \,\text{Scavenging activity}=1-\left(\frac{As-Ab}{Ac}\right)*100$$

whereas: Ab, Ac, and As are the absorbances of the blank (ethanol and sample), the control (DPPH and deionized water), and the sample (DPPH and sample).

Based on comparative in silico analyses, the most potent phytase-producing isolate will be selected for molecular identification and further application.

2.3.4 Molecular identification of the selected isolate

The genomic DNA (gDNA) of the chosen isolate was extracted using a Gene JET Genomic DNA Purification kit, following a modified version of the manufacturer's protocol as detailed by Negm El-Dein et al. [34]. The concentration of DNA was quantified using a Nano-drop device, and the gDNA was preserved at − 20 °C for subsequent analysis. The 16S rRNA gene was amplified via PCR using two specific primers: EuBac-27F and EuBac1492R. The PCR reaction mixture was prepared with PCR Master Mix, primers, and gDNA template. Amplification was carried out in a T100 96-well Thermal Cycler following a specific thermal profile consisting of initial denaturation, multiple cycles of denaturation, annealing, and extension, followed by a final extension step. The quality of the PCR product was evaluated through agarose gel electrophoresis with ethidium bromide staining. The PCR products were then purified using a Gene JET PCR Purification Kit and sequenced using the EuBac-27F primer and an ABI 3130 genetic sequence analyzer. The sequencing process targeted approximately 1500 bp segments covering the V3 region of the 16S rRNA gene sequence. The obtained sequences were compared with accessible sequences in DNA databases using BLAST on NCBI (www.ncbi.nlm.nih.gov/blastn). A phylogenetic tree was constructed using MEGA 11.0 software, employing the neighbor-joining method with bootstrap analysis. The resulting rRNA gene sequences were submitted to the international gene bank.

2.4 Safety assessment for the selected bacterial isolate

2.4.1 Blood hemolysis

The safety assessment of the chosen bacterial isolate focused on its hemolytic activity. To evaluate hemolysin production, actively growing bacterial cells were cultured on Columbia-agar plates enriched with 5% animal blood (rat blood collected from orbital vein). The study was carried out in accordance with the ethical procedures and policies approved by the Animal Care and Use Committee of the National Research Center, Egypt (approval No.: 13050428), which complies with the guidelines from the Canadian Council on Animal Care. The plates were incubated in aerobic conditions at 37 °C for a 24 h period. Aerobic incubation was specifically chosen to avoid potential interference with hemolytic activity that could occur under anaerobic conditions.

After the incubation period, the plates were examined for signs of hemolysis. The hemolytic activity was categorized based on the appearance of zones around the bacterial colonies. A clear zone of hydrolysis indicated β-hemolysis, while a partial hydrolysis zone with a greenish appearance signified α-hemolysis. The absence of any hemolysis was classified as γ-hemolysis [34]. These observations provided insights into the potential safety profile of the bacterial isolate.

2.4.2 Antibiotic susceptibility

The antibiotic susceptibility of the selected bacterial isolate was investigated against the following antibiotics: vancomycin (30 µg/disk), ampicillin (10 µg/disk), amoxicillin-clavulenic acid (20/10 µg/disk), penicillin (10 µg/disk), erythromycin (15 µg/disk), azithromycin (15 µg/disk), sulphamethoxazole-trimethoprim (1.25/23.75 mg/disk), clindamycin (2 µg/disk), doxycycline (30 µg/disk), and linezolid (30 µg/disk) (Bioanalyse limited, Turkey) by disk diffusion method as described by [35]. Briefly, antibiotic discs were placed on inoculated MRS medium with the selected bacterial isolate and incubated at 37 °C for 24 h. According to the inhibition zone diameter, results were interpreted as resistant (inhibition zone less than 5 mm), intermediate resistant (inhibition zone 15 mm or less), or susceptible to the antibiotics (inhibition zone larger than 15 mm).

2.4.3 Histidine and tyrosine decarboxylase activity (histamine and tyramine formation)

Histidine and tyrosine decarboxylase activity was measured as described by Daba et al. [36]. The isolate was streaked in duplicates and incubated for 4 days at 37 °C under anaerobic conditions. Positive histamine and tyramine formation was confirmed when a purple color was observed around the bacterial colonies.

2.5 Immobilization of the selected bacterial isolate

For microencapsulation of the selected bacterial cells, all solutions and equipment should be sterilized at 121 °C for 20 min. Alginate beads were prepared at room temperature according to Bashan [37]. In brief, 250 mL of bacterial culture (24 h old, 109 CFU) was centrifuged, the suspension solution was removed, and the cell pellet was washed with saline solution. It was then suspended in 50 mL of alginate solution (3%) and thoroughly mixed under sterile conditions. This mixture (alginate and cell pellet) was dropped into a sterile calcium chloride solution (3%) using a sterile 5 cm syringe needle, with a distance of 10 cm between the needle and the calcium chloride solution surface. The alginate beads containing bacterial cells were allowed to harden in a calcium chloride solution for 3 h at 4 °C. The supernatant was decanted, and beads were collected, washed with sterile water, and stored in a saline solution for further investigations. The phytase activity of bacteria immobilized in alginate beads was determined by incubating 1 g of the beads in 5 mL of MRS containing phytic acid under the same conditions for phytase production and then measuring the enzyme activity in the surrounding medium. This was repeated several times until the enzyme activity declined.

Phytase synthesis in immobilized cells has been evaluated through several approaches, including measuring enzyme activity after repeated sub-culturing in fresh MRS medium and assessing bacterial growth at OD600. This was usually performed to assess how well the immobilized microorganisms retain their activity over multiple uses, whether it remains functional, and to what extent their activity declines over time. Also, the physical integrity of the alginate beads after multiple cycles was evaluated to see whether the beads maintained their shape and didn’t not disintegrate.

2.6 Statistical analysis

One-way ANOVA was made using Minitab software to indicate the significant difference between treatments at P ≤ 0.05. Principal component analysis (PCA), heat map, and network analyses were performed using Origin Lab, GraphPad Prism, and PAST (4.0) programs, respectively, to visualize the variation of data. The experimental results were expressed as mean ± standard deviation (SD). All statistical analyses were performed using the GraphPad program (GraphPad, San Diego, CA, USA).

3 Results

3.1 Isolation and initial identification of LAB

Different Egyptian samples were used as sources to isolate bacteria (Table 1). A total of fifteen bacterial isolates were isolated as their colonies appeared submerged convex, creamy to whitish creamy on MRS agar medium. Only bacterial colonies that were Gram positive, non-spore formers, non-motile, and catalase negative were selected. As a result, eight isolates were initially identified as LAB. Hence, isolates Nos. 3, 4, 6, 7, 9, 12, 13, and 14 were selected.

Table 1 Sources of bacterial isolates
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3.2 Screening for phytase-producing LAB

The ability of the eight isolates to produce phytase was evaluated through measuring their phytase activity. As shown in Fig. 1, all isolates showed reasonable phytase activities, with the superiority of isolate No. 4. The remaining isolates showed close values; however, those of isolates 14, 7, 3, and 13 were the highest. Hence, isolates 3, 4, 7, 13 and 14 will be chosen.

Fig. 1.
figure 1

Phytase activity (U/mL) of LAB isolates cultured in a modified MRS broth supplemented with 0.725 g/L phytic acid dipotassium salt at 37 °C for 72 h. Data are mean ± SD of two replicates. Different letters are significant at P ≤0.05. R2 = 99.06, P = 0.00.

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3.3 Probiotic characteristics of the selected bacterial isolates

3.3.1 Stress tolerance

The ability to tolerate different stresses was investigated for the five promising phytase-producing isolates. All of the isolates (Fig. 2) displayed promising tolerance to acidic, alkaline, heat, surfactant, and osmotic stress under the conditions mentioned in the materials and methods. Additionally, isolates showed good tolerance to bile salts (at concentrations of 0.05 and 0.1%) and pancreatic enzymes (at 1.5 g/L). Out of all the examined isolates, isolate No. 7 exhibited the least amount of stress response, whereas isolate No. 13 showed the maximum activation upon exposure to various assessed stimuli. The cells from isolates No. 4 and No. 14 had the second-highest stress tolerance, followed by isolate No. 3. On the other hand, isolate No. 3's viability was dramatically activated by alkaline stress, measuring 394.37 ± 0.78% after three h of exposure to pH 9.0. Viability of all tested isolates was totally lost upon exposure to oxidative stress (0.05% H2O2, 30 min) except for isolate No. 4, which was activated and reached 314.81 ± 0.64% (not represented on the figure).

Fig. 2.
figure 2

Stress tolerance (%) of phytase-producing LAB isolates upon exposure to pH stress at pH 2.5 for 3 and 6 h, pH 3.5 for 3 and 6 h, and pH 9.0 for 3 and 6 h, osmotic stress 3.0 M NaCl for 3 and 6 h, heat stress at temperature 55 and 70 °C for 30 and 15 min, respectively, and surfactant stress 0.2% Tween 80, 0.05 and 0.1% bile salts (BS), and 1.5 g/L pancreatic enzymes (PE) for 24 h. Data are mean ± SD of two replicates.

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3.3.2 Cell surface hydrophobicity

The n-hexadecane extraction of active cells was used to measure cell surface hydrophobicity. All isolates were highly hydrophobic (greater than 70%), as shown in Fig. 3 except isolate No. 14, which showed 51.42 ± 0.66% hydrophobicity. The hydrophobicity of the remaining isolates ranged between 78.05 ± 0.94% and 91.60 ± 0.50%. Both isolates No. 3 and No. 4 recorded the highest hydrophobicity % (91.60 ± 0.50%, and 90.82 ± 0.95%, respectively).

Fig. 3.
figure 3

Hydrophobicity (%) of LAB cells. Data are mean ± SD of two replicates. Different letters are significant at P ≤0.05. R2 = 99.82, P = 0.00.

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3.3.3 Antioxidant activity

The in vitro antioxidant activities of cell-free supernatants (CFSs) of the bacterial isolates were evaluated by measuring their DPPH radical scavenging ability. Promising antioxidant activities (rangeing from 77 to 94%) were seen in the CFSs of the chosen bacterial isolates, as illustrated in Fig. 4. The CFS of isolate No. 4 recorded the highest DPPH scavenging activity, measuring 93.57 ± 0.20%, while the CFS of isolate No. 13 achieved the lowest antioxidant activity (76.95 ± 0.051%) compared to the positive control, ascorbic acid, (100%) at the used concentration.

Fig. 4.
figure 4

DPPH radical scavenging activity % of CFSs of LAB isolates. Data are mean ± SD of two replicates. Different letters are significant at P ≤0.05. R2 = 99.33, P = 0.00.

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3.4 In silico comparative analyses for selection of a potent probiotic

As shown in Fig. 5, two principal components (PC1 and PC2) obtained from the property resulted in 70.81% total variance, where PC1 and PC2 accounted for 82.01% and 12.77% of the ten properties, respectively. The projections of the five LAB in the PCA plot were differentiated into two quadrants. Isolates No. 7 and 13 were placed in quadrant II, while isolates No. 3, 4, and 14 were in quadrant IV. LAB in quadrant IV had a greater correlation among their properties with respect to PC1 than other LAB in quadrant II, which showed fewer probiotic properties. Therefore, from the PCA analysis, isolates No. 3, 4, and 14 were selected as the most promising probiotics among others.

Fig. 5.
figure 5

Principal component analysis (PCA) of probiotic properties (acid, alkaline, osmotic, temperature, surfactant, bile, and pancreatic resistances) in addition to cell surface hydrophobicity, antioxidant, and phytase activities of five LAB

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Unlike PCA analysis, the heat map (Fig. 6) visualizes the variation in data by using different colors. When the percentage value increases, the color or intensity of the color varies (from blue to yellow). In this study, as shown in the heat map, isolate No. 4 showed the most potent probiotic, as indicated by the intensity of the yellow color, followed by isolates No. 3 and No. 14, which share almost the same probiotic properties, while isolate No. 13 was a less active probiotic. With no resemblance to any other isolate, isolate No. 7 was the least active probiotic, as seen by the intensity of the blue color. Based on the overall results of heat map, isolate No. 4 was selected as the most promising probiotic due to the almost fully yellow color and intensity. This result was consistent with the result of PCA, which indicated that isolate No. 4 shared strong probiotic properties.

Fig. 6.
figure 6

Heat map plot of probiotic properties (acid, alkaline, osmotic, temperature, surfactant, bile, and pancreatic resistances) in addition to cell surface hydrophobicity, antioxidant, and phytase activities of five LAB

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In Fig. 7, the selected LAB isolates are connected in some ways as network pathways. In contrast to isolate No. 4, which shows better probiotic qualities as shown by node size, isolates No. 3, 13, and 14 had a strong relationship with one another due to somewhat similar probiotic qualities. It was associated only with isolates No. 3, and 14 as indicated by the presence and density of the connecting lines, representing particular similarities. No similarity was indicated between isolate No. 4 and isolates No. 7, and 13, indicating that they have much lower probiotic properties.

Fig. 7.
figure 7

Network analysis plot showing the correlation among five LAB. Node size and line intensity represent the similarity of lactic acid bacteria’s probiotic properties

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In addition, isolates No. 3, and 14 exhibited a comparatively stronger association with the other LAB. However, isolates No. 7, and 13 did not prove strong association with other LAB. From the network analysis, isolate No. 4 was selected as the most promising probiotic, as confirmed also with PCA and heat map analyses.

3.5 Molecular identification of the selected isolate

The highest homology obtained for the DNA sequence of the 16S rRNA gene corresponding to position 8–1492 of the selected isolate No. 4 was to sequences of P. pentosaceus strain HM75-1 16S ribosomal RNA gene, partial sequence, and to P. acidilactici strain 2224 16S ribosomal RNA gene, partial sequence (99.8% identity). As a result, the sequence was deposited in the gene bank as P. pentosaceus strain NMP4762Ch under the accession number MZ413646.1. On the other hand, a phylogenetic tree was constructed (Fig. 8) by MEGA 11.0 software using neighbor joining and the Bootstrap method using a number of 1000 bootstrap replications.

Fig. 8.
figure 8

Neighbor-joining phylogenetic tree constructed using MEGA 11.0 displays the position of Pediococcus pentosaceus strain NMP4762Ch (black diamond) among other Pediococcus strains based on 16s rRNA sequences. As outgroups, the sequence of Enterococcus faecalis ATCC 19433 (AB012212) and Aerococcus viridans ATCC 11563 (M58797) were used. The scale bar represents 0.02 nucleotide substitutions per site

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3.6 Safety assessment of the identified strain

3.6.1 Hemolytic activity

In terms of hemolytic activity, P. pentosaceus showed no clear or green zones on the blood plates, indicating that it has no hemolytic activity.

3.6.2 Antibiotic susceptibility

P. pentosaceus's susceptibility to 10 distinct widely-used antibiotics was evaluated. It displayed resistance to vancomycin, ampicillin, sulphamethoxazole-trimethoprism, amoxicillin-clavulanic acid, and ampicillin as indicated by clear zones less than 5 cm. However, it demonstrated susceptibility to penicillin, linezolid, clindamycin, erythromycin, and doxycycline, with reported inhibition zones of 34, 29, 22, 20, and 16 mm, respectively, and was only moderately resistant to azithromycin (inhibition zone 15 mm).

3.6.3 Histamine and tyramine formation

In the screening media employed, P. pentosaceus did not exhibit any activity of histidine or tyrosine decarboxylase synthesis, indicating that it was unable to create the biogenic amines, histamine or tyramine.

3.7 Immobilization of P. pentosaceus NMP4762Ch cells and quantitative determination of phytase activity

The reusability of P. pentosaceus NMP4762Ch cells for continuous phytase synthesis has been evaluated in the immobilized phytase-producing cells. As illustrated in Fig. 9, immobilization in alginate beads had no effect on the amount of phytase produced. After 14 days, the highest amount of phytase (432.0 ± 35.4 U/mL) was produced; after that, it began to progressively decrease until it reached 142.5 ± 10.6 U/mL after 56 days. Conversely, the maximum shelf stability was observed at 557.5 ± 9.5 U/mL after 14 days; however, this value declined throughout time, peaking at 133.5 ± 3.5 U/mL by the end of the 56th day.

Fig. 9.
figure 9

Reusability and shelf-life stability of the immobilized Pediococcus pentosaceus strain NMP4762Ch as indicated by phytase (U/mL)

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The growth of the immobilized cells remained active as indicated by their consistent OD600 measurements throughout the experiment, with no decline observed.

Also, the physical integrity of the alginate beads was evaluated, as they maintain their shape and do not disintegrate after multiple cycles.

4 Discussion

LAB are promising biotechnological tools that have contributions in various pharmaceutical, industrial, and food-related fields. It is utilized not only for food preservation and fermentation but also for boosting the bioavailability of multivalent positive cations that are chelated by phytic acid in food. Because of their capacity to produce phytases that hydrolyze phytic acid by releasing phosphate groups one at a time, they are able to increase the ionic and soluble content of various metal ions. These solubilized minerals can therefore be assimilated and absorbed by humans and animals’ bodies and become bioavailable. Additionally, as phytate hydrolyzes, numerous myo-inositols are released as byproducts. These myo-inositols are crucial for controlling and regulating a range of metabolic processes [38]. Therefore, LAB-encoding phytases are considered a promising starter culture suitable for legume and cereal fermentations [39].

In this investigation, eight of the fifteen bacterial isolates that were acquired from various Egyptian sources were initially identified to be LAB. The isolates No. 3, 4, 7, 13, and 14 were the most effective in producing phytase among them.

There have been prior reports of phytase activity by LAB from fermented food [38, 40], particularly for phytases released by LAB isolated from sourdough [20, 21]. Intracellular phytase activity has been reported by De Angelis et al. [41], with particular reference to isolates of Lactobacillus sanfranciscensis. However, Doğan and Tekiner [22] have reported extracellular phytase activity of a large number of LAB. By investigating phytase production among the presumptively identified LAB, five isolates (No. 3, 4, 7, 13, and 14) were chosen, with isolate No. 4 being the most promising. This isolate yielded a higher phytase (411 U/mL) than either Pediococcus pentosaceus KTU05-8 or KTU05-9 strains, which could produce extracellular phytase of about 32 and 54 U/ml, respectively, using phytic acid dipotassium salt [21].

Probiotics are distinguished by their capacity to tolerate stress conditions comparable to those observed throughout the gastrointestinal tract, such as the high acidity found in the stomach, and alkalinity in the intestine, in order to fulfill their nutritional and therapeutic roles [42]. Probiotics should also be able to withstand oxidative and osmotic stressors found in the gastrointestinal tract, as well as endure and multiply at physiological levels of bile salts [43, 44]. It's interesting to indicate that the stress tolerance capabilities of the five phytase-producing organisms under investigation showed that they could not only tolerate heat stress (55 and 70 °C for 15 min), but also exposure to high temperatures activated their viability. This was also noted for the other stress conditions. De Angelis and Gobbetti [45] discussed the mechanism of heat tolerance in some LAB genera and described it as a multiple-step process. Also, they described the influence of subjecting exponential phase cells to 50 °C for 30 min, which resulted in an increase in the viability that can reach 1000-fold, depending on the strain. However, Jonathan et al. [46] reported that P. acidilactici B14's viability decreased after being exposed to 60 °C for more than 5 min. They also noted that P. acidilactici was not affected by heat treatment that ranged from − 80 °C to 60 °C for 5 min.

Gastric juice has a pH of 1.5–3.5; therefore, it's important to determine whether probiotic candidates can survive in low-pH conditions [47]. Low pH levels in the gastric juice damage the bacteria's cell wall and membrane, which affects the membrane pathway and causes unfavorable metabolic processes, energy depletion, and ultimately cell death [48]. The current study's tested isolates demonstrated a promising tolerance to acidic and alkaline pH stress (pH 2.5, 3.5, and 9) for durations of three and six h. Depending on their phenotypic traits and the surrounding conditions, many LAB can survive at low pH [49]. These LAB may thrive and multiply in severe gastrointestinal environments; therefore, their tolerance to low pH is crucial.

The tested isolates, with the exception of isolate No. 4, totally cease to exist after 30 min of exposure to oxidative stress (0.05% H2O2). In accordance with De Angelis and Gobbetti [45], the various types and amounts of antioxidative mechanisms that some LAB genera employ determine their capacity of withstanding oxidative stress. Regarding osmotic stress, viability of cells was also activated after treatment with 3 M NaCl for 3 and 6 h. Tolerating the presence of high concentrations of sodium chloride is an advantage, especially if these isolates will be involved in food applications.

On the other hand, MRS was supplemented with 0.2% Tween 80 to induce detergent stress. As a result, all tested isolates succeeded in growing under such stress. This can be attributed to the vital incorporation of Tween 80 into the cell membrane during growth. It should be noted that Tween 80 (also known as Polysorbate 80 or E433) is a common food additive that is used in food processing [34]. Moreover, Tween 80 can enhance tolerance to bile salt and contribute to improving survival and adhesion of starters with cholesterol uptake potential [50, 51].

Exposing to bile salts (as the case in the gastrointestinal tract) has a negative impact on phospholipids in the cell membranes of bacterial cells, resulting in disruption of their cellular homeostasis. Therefore, withstanding bile is critical for the survival and subsequent colonization of probiotics inside the gastrointestinal tract [36]. All isolates tolerated bile salts after sub-culturing in elevated concentrations of bile salts (up to 0.1%). These results came in accordance with those achieved by P. acidilactici B14, which tolerated exposure to 0.3% bile salts [46]. Similarly, isolates under investigation tolerated pancreatic enzymes.

As revealed from the aforementioned results of probiotic properties and phytase activity, the results of the 5 isolates were overlapping, and it would be difficult to select the most promising probiotic without the aid of the statistical analyses; therefore, in silico comprehensive analyses based on the construction of PCA, heat map, and network analyses were made that confirmed the selection of isolate No. 4 as the most potent phytase-producing probiotic. This isolate was identified via 16S rRNA sequencing as P. pentosaceus strain NMP4762Ch and deposited in the gene bank under the accession number MZ413646.1.

The safety of this strain was investigated in terms of its antibiotic susceptibility, hemolytic activity, and ability to produce histamine and tyramine as one of the critical criteria for choosing probiotic bacteria [52]. It showed no hemolytic activity and was resistant to vancomycin, which is an intrinsic feature among many LAB genera such as Leuconostoc, Lactobacillus, and Pediococcus [53]. Also, it was resistant to the tested concentrations of ampicillin, amoxicillin-clavulanic acid, and sulphamethoxazole-trimethoprism.

On the other hand, P. pentosaceus strain NMP4762Ch didn’t produce histamine and tyramine, which can be considered a probiotic safety character because biogenic amines are known to have adverse impacts on human health [54]. Histidine decarboxylase activity was reported among some LAB species [55, 56]. Therefore, the incapability of the selected strain to create histamine and tyramine is an advantage, especially if this strain will be employed in the production of functional products.

Big data sets are becoming more common and can be challenging to comprehend. Principal component analysis (PCA) is a technique used to reduce the dimensionality of datasets, enhance interpretability, and minimize information loss [57].

The selection of an effective probiotic candidate was accomplished by employing principal component analysis (PCA), heat map, and network analysis. Ten factors were considered in this process, including acid, alkaline, osmotic, temperature, surfactant, bile, and pancreatic stresses. Additionally, the hydrophobicity of the cell surface, antioxidant activity, and phytase activity were taken into account. PC1 accounts for the majority of the variation in the property data, while PC2 accounts for the remaining variation. PC2 is orthogonal to the initial PC1 [58]. Principal Component Analysis (PCA) identified isolates No. 3, 4, and 14 as the most favorable probiotics compared to the rest.

After analyzing the heat map, isolate No. 4 was chosen as the most favorable probiotic due to the almost fully yellow color and intensity. This finding was consistent with the result of PCA, which suggested that isolate No. 4 exhibited significant probiotic characteristics. Network analysis aids in verifying the similarity of LAB characteristics by visually representing the level of confidence in similarity and the color of the nodes. Typically, the strains that are being researched are represented as nodes, and the size of each node is directly proportionate to the number of connections. Furthermore, the density of the connecting lines is indicative of the magnitude of their similarities [59]. All three techniques (PCA, heat map, and network) yielded comparable results in identifying potential probiotic candidates. The in silico analyses were used to precisely compare and analyze the results, ultimately leading to the selection of isolate No. 4 as the most promising probiotic. These computational analyses have verified that the use of PCA, heat map, and network analyses may reliably assist in the accurate selection of the most powerful candidate that possesses the attributes required from the study. In the previous research, the most promising probiotics from seven LAB were identified as L. casei IDCC3451, L. plantarum LP-K1791, and L. rhamnosus IDCC3201 by the use of in silico analyses, which included principal component, heat map, and network analyses. The findings of Mallapa et al. [60] were supported by the identical and contradicting results from PCA and heat map studies, which showed that heat map analysis, as opposed to PCA analysis, allowed them to choose nine Lactobacillus isolates from a group of fourteen probiotic isolates. Subtractive screening was carried out on 16 cultures using several tests, including the rate of acidification and the growth of LAB upon the addition of fructooligosaccharide (0–0.45 mM) and inulin (0–20 mM). Following principal component analysis (PCA) of several strains using prebiotic substrates at varying doses, Lactobacillus paracasei CD4 was chosen as a promising strain [58].

The genus Pediococcus is a famous LAB member that is commonly used as a starter culture due to its proteolytic and acidifying activities, which have positive impact on the sensory properties of fermented products [61]. Also, some Pediococcus strains are known as famous probiotics and bacteriocin-producing bacteria [62, 63]. Cizeikiene et al. [21] showed that the highest extracellular phytase activity was produced by P. pentosaceus strains from rye sourdough with 32 to 54 U/mL, respectively, under conditions similar to leavening of bread dough. On the other hand, out of 21 phytase extra producing LAB, the average extracellular phytase activity was 656.8 ± 188.1 U/mL, and a P. pentosaceus EK1 isolate showed the highest activity as 1285.5 U/mL [22].

Generally, using alginate gel beads for immobilization of the whole cells has been described as an efficient method to increase enzyme activity. Moreover, it facilitates separation of products, ensures the long-life activity of enzymes, and reduces chances of cell contamination [64]. A previous study has described immobilization of Candida krusei cells in calcium alginate for efficient production of phytase [65]. Similarly, immobilizing Saccharomyces cerevisiae cells in alginate beads resulted in phytase activity and immobilization yield that reached 280 mU/g-bead and 43%, respectively [66]. P. pentosaceus strain NMP4762Ch was immobilized in alginate beads for continuous production of phytase. The impact of immobilization on reusability and shelf-life stability revealed that the cells were still alive for over two months with the ability to continuously produce phytase reaching its maximum production after 14 days and declining gradually till the end of the next month. This was challenging because LAB are normally non-spore formers and couldn’t be alive on the shelf for more than two weeks.

5 Conclusion

Phytic acid in cereals reduces nutrient bioavailability by binding to minerals and proteins, leading to micronutrient deficiencies. Phytases, enzymes that break down phytate, are essential keys for resolving this concern. Lactic acid bacteria (LAB) are safe, versatile microorganisms known for producing various enzymes, including phytases. This study evaluated the probiotic potential of five phytase-producing LAB strains. Through comparative in silico analyses, P. pentosaceus strain NMP4762Ch was identified as the most promising candidate. The strain was immobilized on alginate for continuous phytase production. Incorporating this phytase-producing probiotic into economical diets, particularly for diabetic and obese individuals, could improve nutrient bioavailability from cereals and by-products like wheat bran, potentially reducing the risk of anemia and osteoporosis associated with long-term consumption of such diets.