INTRODUCTION:

In nature, probiotics are widely distributed^1,2. Foods containing probiotics are consumed by people in many areas³, and these foods have been shown to play significant roles in maintaining human health. Prebiotics, on the other hand, have been extensively researched recently as they are less risky and easier to market than probiotics^4,5. Prebiotics can improve the function of the epithelial barrier^6,7, modulate the gut microbiota in a positive way^8–10, boost immunological responses, and affect systemic metabolism^10,11.

Additionally, cell-free supernatants, exopolysaccharides (EPS), enzymes,cell wallscopmonents, and bacterial extracts are the main subjects of prebiotic research.

EPS, a polysaccharide with a long chain of high molecular weight, has a broad range of forms and physicochemical characteristics¹². These polysaccharides are released into the extracellular space and are secondary metabolites made by several bacteria, including Lactobacillus and Bacillus^13,14. When EPS is contrasted with other prebiotics, it has numerous benefits. Cost-effectiveness: a. because the medium needed to produce extracellular polymer shells (EPS) is inexpensive, and b. because EPS is secreted into the extracellular space, the extraction process is straightforward; c. the type and the composite of EPS are dependent on the species of microbes and the production medium¹⁵.

Theoretically, a genus grown in various media conditions could yield EPSs with distinct structural variations. This offers EPS screening with beneficial functions a quantitative foundation. For example, EPS is amphiphilicwith the ability to function as a natural emulsifier due to the hydrophobic group connected to its hydrophilic chain. Low-fat mayonnaise's rheological qualities and stability may be enhanced by adding EPS ^16,17, and some EPSs' emulsification activity was shown to be on par with xanthan gum. Furthermore, due to EPS's superior antioxidant capability, it might be utilized as a possible antioxidant in the food processing industry¹⁸. As a result, there is a lot of potential for using these EPS features as basic materials for functional food additives.

Further advantage of EPS;theweight of EPS is the first. Numerous factors, including the types of the microorganisms, the medium's components (such as a carbon or nitrogen source), and the specific nutrients that each type of microorganism prefers for EPS formation, all have an impact on the creation of EPS^13,19. Also, the biosynthesis of EPS is also influenced by the culture conditions, such as temperature and pH^20,21. Furthermore, the growth of microorganisms is not always compatible with the ideal circumstances for EPS generation. Microorganisms can only synthesize some EPS when they are under stress²². While 37°C is the ideal temperature for Lactobacillus paracasei development, 20°C is the ideal temperature for producing EPS²⁰.

To maximize the EPS yield, these elements must be optimized systematically. The biological activity of the EPS is the other issue. The path of research and application is determined by the function, whereas the yield of EPS establishes its industrial viability. Owing to the variety of EPS structures, EPS has a wide range of actions, including immunomodulatory, antioxidant, anti-inflammatory, emulsification, antibacterial, anti-tumor, and cholesterol-lowering properties^23,24. For this reason, EPS has to have its possible biological activities identified in order to be further investigated and used in the food sector.

Rather than necessitating a greater number of tests, which are frequently expensive and time-consuming, the design was utilized to change the interaction of numerous variables and their interaction in any one experiment in one-step^25-27. Our goal is to investigate how well the Taguchi experimental design can optimize and evaluate the variables influencing the probiotic Lactobacillus brevis ability to produce extracellular polymer (EPS).

MATERIALS AND METHODS:

Probiotic strain:

The Lactobacillus brevis strain was maintained in an MRS medium with 50% glycerol at -80°C. The turbidity of bacterial growth was measured using the 0.5 McFarland standards, which involved utilizing a standard inoculum of 100 microliters (1x10⁷cells/ml) of bacterial growth culture and 48 hours of static incubation at 30°C.

EPS production and extraction:

EPS was produced by propagating the Lactobacillus brevis culture in MRS broth medium, containing g/l: D(+) glucose, 20.0; peptone, 10.0; meat extract, 8.0; yeast extract, 4.0; K₂HPO₄, 2.0; CH₃COONa · 3H₂O, 5.0; triammonium citrate, 2.0; MgSO₄ · 7H₂O, 0.2. pH 6.2 ²⁸. With slight modifications, the EPS was extracted and purified in accordance with Cerninget al.²⁹. The cells were extracted by centrifugation at 8000 rpm for 5 min at 4°C after the growing cultures were heated to 100 °C for 5 min. Ethanol 100% 2vol were used to precipitate the EPS. The precipitate was appeared by centrifuged at 8000rpm for 20 minutes after standing overnight at 4°C. Following a 24hour dialyzation against deionized water at 4°C, the EPS was dissolved in deionized water and freeze-dried. To extract the proteins, the freeze-dried powder was treated in 10% (w/v) trichloroacetic acid. After five days of dialyzing at 4°C against deionized water, the supernatant was freeze-dried. These preparations were kept in storage at 4°C and were known as pure EPS.

Optimization conditions of EPS production:

In order to get a reliable result, the Taguchi technique was developed gradually, involving the identification of crucial factors, the construction of an accurate matrix, the statistical analysis of the data, and validation using the best values. EPS dry weight ultimate yield and biosynthesis rate were assessed using this design in multiple studies. The optimal conditions for optimizing EPS dry weight were determined in these qualitative and quantitative screening tests by analyzing biosynthesis reaction ingredients, such as glucose (%), pH, temperature (°C), incubation time (h), inoculum size (µl), and volume (ml) from tube 20 ml, among other biosynthesis reaction parameters. The L₂₇(3⁶) Taguchi orthogonal array design with six factors, three levels, and 27 runs was chosen for this optimization method (Table 1). The average of produced EPS for each process condition as intended is calculated using the MINITAB 18 program, and then the F test and ANOVA wasused to examine the importance of all components and their correlations at particular levels. Ultimately, the results generated by applying Taguchi's method were compared to the real experimental value in a confirmation test^6,10,30.

Table 1: Taguchi design Factors and levels using for EPS production

Factors	Level 1	Level 2	Level 3
pH	4	6.5	8
Temp.°C	20	30	40
Incubation time (h)	12	24	48
Inoculum size (µl)	50	100	150
Volume (ml)	5	10	15
Glucose (%)	1	2	3

RESULTS AND DISCUSSION:

The design of a biotechnological process could optimize the pH of the Lactobacillus brevis culture, temperature (°C), incubation time (h), inoculum size (µl), volume (ml), and glucose (%) to achieve maximum production.

With the goal to maximize the production of EPS by the Lactobacillus brevis's strain, an integrated optimization of glucose transformation processing parameters was used for the first time in this work., Taguchi experimental design was used to adjust parameters on a small scale.

Taguchi's L₂₇(3⁶) orthogonal array design was utilized in order to optimize the strain Lactobacillus brevis's EPS weight (mg/L). To find the generated EPS weight values, 27 trials using six components categorized according to the L₂₇ (3⁶) OA design were conducted, as Table 2 illustrates. The ANOVA and F-test can be used to assess the experimental data, as shown in Table 3. Our model's R2 of 40.60% showed that the experimental data suited the model well. Both the model and its parameters had a high degree of significance (P < 0.5). Table 3 shows that the modified R2 value of 22.78% was reasonably close to the expected R2 value of 0.0 %.

Table 2: The experimental design and response results

Run	pH	Temp.	Incubation time (h)	Inoculum size (µl)	Volume (ml)	Glucose (%)	Response (mg/L)
1	4.0	20	12	50	5	1	93
2	4.0	20	12	50	10	2	117
3	4.0	20	12	50	15	3	0
4	4.0	30	24	100	5	1	370
5	4.0	30	24	100	10	2	390
6	4.0	30	24	100	15	3	0
7	4.0	40	48	150	5	1	265
8	4.0	40	48	150	10	2	287
9	4.0	40	48	150	15	3	0
10	6.5	20	24	150	5	2	560
11	6.5	20	24	150	10	3	0
12	6.5	20	24	150	15	1	210
13	6.5	30	48	50	5	2	420
14	6.5	30	48	50	10	3	0
15	6.5	30	48	50	15	1	230
16	6.5	40	12	100	5	2	180
17	6.5	40	12	100	10	3	0
18	6.5	40	12	100	15	1	120
19	8.0	20	48	100	5	3	0
20	8.0	20	48	100	10	1	0
21	8.0	20	48	100	15	2	32
22	8.0	30	12	150	5	3	0
23	8.0	30	12	150	10	1	0
24	8.0	30	12	150	15	2	42
25	8.0	40	24	50	5	3	0
26	8.0	40	24	50	10	1	0
27	8.0	40	24	50	15	2	76

Table 3: The ANOVA analysis of the means variance

Source	DF	Adj SS	Adj MS	F-Value	P-Value
Regression	6	276133	46022.1	2.28	0.077
pH	1	81622	81622.2	4.04	0.058
Temp.	1	392	392.0	0.02	0.891
Incubation time (h)	1	14684	14684.5	0.73	0.404
Inoculum size (uL)	1	10177	10176.9	0.50	0.486
Volume (mL)	1	77094	77093.6	3.82	0.065
Glucose (%)	1	92164	92163.6	4.56	0.045
Error	20	404028	20201.4
Total	26	680160

Figure 1 shows the major effects plot for the mean EPS based on Taguchi's experimental results. The standardized estimated impacts of the many variables evaluated in the prospective experiment on the production of EPS are shown by the Pareto graph in Figure 2. Figure 3: A histogram of the residual values that displayed the frequency of each value interval against the residual values; b) A random scattering of the residuals versus the fitted values; and c) A normal probability plot of the standardized residuals for the production of EPS, where the probability was linear; d) An asymmetrical pattern in the residuals versus the observation order. Figure 4: Response surface plots for exopolysaccharide yield. The P-values indicated that the culture's pH, volume (ml), and glucose percentage (%) were important variables. Finally, a confirmation test for this design is required to verify the proper parameters for enhancing EPS output. Twenty seven of trials were carried out under these circumstances, and the optimal concentrations for each factor were determined. The outcome of the confirmation experiment is compared to the expected value based on the variables and their measured levels, indicating a tight relationship between the experimental and projected values. It is possible to refine the approach and raise the EPS weight. Using the Taguchi in the biosynthetic EPS pathway, the synthesis of EPS increased to 560mg/L from the basal condition of 376mg/L.

Response surface methodology (RSM) was used by Wang et al.³¹ to improve the primary medium components and culture conditions that influence Lactobacillus plantarumR301's synthesis of extracellular polymer shells (EPS). The maximum EPS yield rose by 84.70%, from 53.34mg/L prior to optimization to 97.85mg/L following optimization.

According to all responses, Ermişet al. ³² determined that the ideal incubation conditions for Lactobacillus brevisE25 were 35 C, 18 hours, and an initial pH of 6.5. This produced an average generation of EPS that ranged from 10 to 35g/L. A decrease in EPS yield over prolonged incubation periods may be related to the fact that EPS is produced as primary metabolites, which are growth-associated.

The ideal medium composition by RSM, according to Choi et al.³³, was found to consist of 31.29 maltose, 30.27g/l yeast extract, 39.43 soytone, 5 sodium acetate, 2 K₂HPO₄, 1 Tween 80, 0.1 MgSO₄.7H₂O, and 0.05 MnSO₄.H₂O(g/l). The maximal biomass was estimated to be 3.951g/l. Lactobacillus plantarum200655's biomass under the optimized medium was 3.845g/l, 1.58 times greater than that of the unoptimized medium (2.429g/l) and comparable to the expected value.

Figure 1: The main effects plot of EPS mean results in Taguchi’s experimental

Figure 2: Pareto graph showing the standardized estimated effects of the many factors examined in the potential experiment on the generation of EPS

Figure 3: The EPS production residuals were plotted as a normal probability plot, and the probability indicated linearity, The residuals versus fitted were randomly distributed, The residuals versus residual values histogram displayed the frequency of each value interval versus the residual values, The residuals versus observation order exhibited an asymmetrical pattern

CONCLUSION:

Applying the Taguchi experiment, we were able to produce EPS at a higher ratefrom Lactobacillus brevis in a fewer time, resources, and experimental errors. The statistically significant variables were then determined by an examination of the standard variance approach. Since each factor (6) had three levels, the L-27 Orthogonal Array was chosen for the experimental design. The following parameters yielded the best test results: pH of the Lactobacillus brevis culture (6.5), temperature, length of incubation (24hours), inoculum size (150µl), volume (5ml), and glucose (2 %). the mean impacts of the relevant interactions between the influencing parameters at the specified levels on the synthesis of EPS. The EPS weight can be increased and improved approach. The synthesis of EPS (560mg/L) increased using the Taguchi in the biosynthetic EPS pathway as compared to the basal condition (376mg/L).

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Received on 07.06.2024 Revised on 14.09.2024

Accepted on 21.11.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5803-5808.

DOI: 10.52711/0974-360X.2024.00882