Bacteriocin Production by Lactiplantibacillus plantarum LD1 in Solid-State Fermentation Using Lignocellulosic Substrates

Abstract

In this study, solid-state fermentation for growth and bacteriocin production by Lactiplantibacillus plantarum LD1 was carried out using wheat bran, a lignocellulosic substrate. This is the first report showing bacteriocin production using L. plantarum LD1 in solid-state fermentation. Wheat bran supported higher production of bacteriocin (391.69 ± 12.58 AU/mL) than other substrates. Appropriate conditions were achieved using statistical designs. Significant factors identified by Plackett–Burman Design and their interactions were studied using response surface methodology. Enhanced production of bacteriocin (582.86 ± 0.87 AU/mL) and optimal growth (log₁₀ CFU/mL 8.56 ± 0.42) were attained in wheat bran medium supplemented with peptone (1.13%), yeast extract (1.13%), glucose (1.56%), and tri-ammonium citrate (0.50%). Growth in non-optimized medium (MRS) was almost similar (log₁₀ CFU/mL 8.15 ± 0.20), but the bacteriocin production level was lower (391.69 ± 0.58 AU/mL). Bacteriocin production was sustainable using varied quantities of wheat bran, showing the suitability of the optimized bioprocess for large-scale production. The cost for bacteriocin production in the optimized medium was found to be 444,583.60 AU/USD, which is about 4 times more economical than the cost of the commercial MRS medium, 121,497.18 AU/USD). Thus, an almost 1.5-fold improvement in bacteriocin production was achieved using wheat bran as the substrate. The cost of the production medium was reduced by approximately 25%, making the bioprocess economical.

1. Introduction

Lignocellulosic substrates such as rice straw, wheat bran, corn cobs, sugarcane bagasse, etc., are excellent options for various bioprocessing applications due to their abundance, cost-effectiveness, and richness in cellulose, hemicellulose, and lignin. When these substrates are supplemented with additional nutrients, they create an environment conducive to the growth and metabolism of bacterial strains, thereby improving bioprocess efficiency and yield [1,2]. The growth of microbes in such matrices in the absence or near absence of water and free-moving air is known as solid-state fermentation (SSF). In recent years, interest in the growth of beneficial bacteria under SSF conditions has increased due to its economical nature and because it provides simulated conditions similar to those in the gut and solid foods, where probiotics generally grow [3]. The growth conditions such as humidity and temperature in SSF resemble the natural environment, and it is an environmentally friendly bioprocess for the revalorization of waste to produce novel microbial metabolites [4,5].

Wheat bran is one of the most commonly used matrices for SSF due to its low cost and availability on farms and in the milling industry. It contains about 53% dietary fiber (xylan, galactan, lignin, fructan, and cellulose), 8–12% moisture, 9–18% proteins, 4–8% ash, 60–75% carbohydrates, and 10–30% starch, which promotes the growth of probiotic lactic acid bacteria (LAB) [6,7]. It also contains anti-nutritional compounds such as phytic acid (inositol polyphosphate) and tannins [8]. LAB increase the nutritional value of wheat bran after fermentation and the degradation of anti-nutritional compounds [9,10].

Some LAB species are the most popular probiotics, classified as Qualitative Presumption of Safety (QPS) and Generally Recognized as Safe (GRAS) due to their nonpathogenic behavior and safe applications in humans and animals [11,12]. In general, these bacteria are non-motile, Gram-positive, rod- or cocci-shaped, and have the capability to ferment carbohydrates and produce antimicrobial compounds like organic acids, acetoin, antibiotics, diacetyl, hydrogen peroxide, and bacteriocins [13,14]. Bacteriocins are proteins produced by most LAB and can be used as antimicrobial alternatives to chemical preservatives in food safety and clinical antibiotics in therapeutics [15,16].

Appropriate growth conditions can be achieved using different methods, such as the one-variable-at-a-time (OVAT) approach and statistical designs. Plackett–Burman Design (PBD) and response surface methodology (RSM) are the most commonly used statistical designs to identify important variables and analyze their interactions [17]. For the growth of probiotic bacteria and the production of bacteriocins, commercial media are generally used, which are too expensive and consist of complex nutrients which interfere with the purification of bacteriocins and other metabolites. Thus, there is a need to develop alternative cost-effective culture media suitable for microbial growth and bacteriocin production in SSF conditions.

Lactiplantibacillus plantarum LD1, isolated from an indigenous fermented food, Dosa batter, has been characterized for its probiotic properties in our laboratory [18]. The bacteriocin produced by L. plantarum LD1 inhibited several pathogenic bacteria, suggesting its potential application in food safety and therapeutics [19]. In this study, the growth of L. plantarum LD1 on wheat bran was investigated as a matrix for the low-cost production of bacteriocins for industrial applications.

2. Materials and Methods

2.1. Raw Materials, Growth Conditions, and Bacterial Strains

Various agricultural residues such as wheat bran, rice bran, mustard oil cake, sugarcane bagasse, corncobs, congress grass, rice straw, oat bran, and chickpea bran were collected from local markets (

Table 1. Growth, final pH, and bacteriocin production of Lactiplantibacillus plantarum LD1 on different substrates moistened with MRS medium.

S. No.	Substrates	Growth (log10 CFU/mL) *	Final pH *	Bacteriocin Production (AU/mL) *
1	Wheat bran	8.15 ± 0.20	4.64 ± 0.07	391.69 ± 12.58
2	Rice straw	7.66 ± 0.05	5.17 ± 0.06	219.80 ± 8.58
4	Mustard oil cake	7.43 ± 0.05	4.84 ± 0.04	362.50 ± 22.50
5	Sugarcane bagasse	8.43 ± 0.13	4.92 ± 0.20	284.56 ± 17.50
6	Corn cobs	7.92 ± 0.03	4.68 ± 0.01	10.89 ± 0.21
7	Congress Grass	7.69 ± 0.37	5.15 ± 0.03	4.68 ± 0.12
8	Rice bran	8.09 ± 0.09	4.78 ± 0.19	380.73 ± 18.29
9	Oat bran	7.74 ± 0.24	5.54 ± 0.12	20.54 ± 0.20
10	Chickpea bran	6.16 ± 0.09	5.72 ± 0.12	17.12 ± 0.13

* Values are expressed as the mean of three independent experiments with ±S.D.

). All substrates were washed three times with distilled water to remove excess starch and dust, and then dried at 60 °C in a hot air oven (Narang Scientific Works India Limited, Gurugram, India) and ground using a grinding mixer. The ground matrices were used as solid substrates for the growth of L. plantarum LD1 under SSF conditions.

L. plantarum LD1 was grown in the presence of different substrates moistened with MRS medium (1% peptone, 1% meat extract, 0.5% yeast extract, 2% glucose, 0.1% tween 80, 0.2% tri-ammonium citrate, 0.5% sodium acetate, 0.05% magnesium sulphate, 0.02% manganese sulphate, or 0.2% di-potassium phosphate and pH 6.8) at 37 °C for 18 h in a Bio-chemical Oxygen Demand (BOD) incubator (Bipro Scientific Instruments, New Delhi, India). For bacteriocin activity tests, Micrococcus luteus MTCC 106 was used as the indicator strain and grown in Nutrient Broth (NB) medium (0.5% peptone, 0.3% meat extract, 0.5% NaCl, and pH 7.0 at 37 °C) at 200 rpm in a BOD shaker incubator (Scigenics Biotech, Chennai, India) for 18 h, as previously described [18].

2.2. Growth and Production of Bacteriocin

An amount of 5 g of each substrate was moistened with 10 mL of MRS medium and sterilized using an autoclave (121 °C at 15 psi for 15 min). The freshly grown culture of L. plantarum LD1 (~10⁶ CFU/mL) was inoculated in each substrate and incubated at 37 °C for 24 h. The growth in terms of log₁₀ CFU/mL, acid production in terms of reduction in pH, and bacteriocin production in terms of activity unit per mL (AU/mL) were monitored in each set of substrates used, as suggested by Cotarlet et al. [20].

To determine log₁₀ CFU/mL, 1 g of the respective matrix was mixed with 10 mL of normal saline (0.8% NaCl) and 10-fold serially diluted. An aliquot of 100 µL was plated on MRS agar medium for the appearance of colonies of L. plantarum LD1, which were counted manually, and CFU/mL was calculated using the following formula: CFU/mL = Number of colonies/Dilution factor ×Volume used.

The production of acids during the growth was monitored in terms of reduction in pH using a pH meter (Systronics, New Delhi, India). For the extraction of bacteriocin, the fermented substrate was mixed with 5 mL distilled water and incubated at 200 rpm for 2 h. The mixture was filtered with muslin cloth, and the filtrate (5 mL) was further centrifuged at 10,000 rpm for 10 min to collect the cell-free supernatant (CFS), as suggested by Tong et al. [21]. The CFS was tested for antimicrobial activity against the indicator strain M. luteus MTCC106 using the agar well diffusion assay (AWDA), as performed previously [18]. Thereafter, the diameter of the clear zone of growth inhibition around the wells was measured and used for the calculation of bacteriocin activity in terms of activity unit per mL using following formula [22]:

AU/mL = Area of clear zone − Area of well/Volume loaded in the well

2.3. Bioprocess Development Using Plackett–Burman Design

The culture conditions were statistically optimized using Plackett–Burman Design (PBD) to select important variables. The experimental design using eleven selected independent variables was performed as depicted in

Table 2. Growth, final pH, and bacteriocin production of Lactiplantibacillus plantarum LD1 using Plackett–Burman Design.

Run	Peptone (%)	Meat Extract (%)	Yeast Extract (%)	Glucose (%)	Sodium Acetate (%)	Tri-Ammonium Citrate (%)	Di-potassium Phosphate (%)	Manganese Sulphate (%)	Magnesium Sulphate (%)	Tween 80 (%)	Substrate	Growth (log10 CFU/mL) *	Final pH *	Bacteriocin Production (AU/mL) *
1	0 (−)	0 (−)	0.5 (+)	2 (+)	0.5 (+)	0 (−)	0.2 (+)	0.02 (+)	0 (−)	0.1 (+)	WB	6.31 ± 0.27	4.82 ± 0.16	1.89 ± 0.01
2	1 (+)	0 (−)	0 (−)	0 (−)	0.5 (+)	0.2 (+)	0.2 (+)	0 (−)	0.05 (+)	0.1 (+)	WB	7.95 ± 0.38	5.30 ± 0.03	11.22 ± 0.12
3	1 (+)	1 (+)	0 (−)	2 (+)	0.5 (+)	0 (−)	0.2 (+)	0 (−)	0 (−)	0 (−)	WB + RS	7.66 ± 0.29	4.56 ± 0.18	22.13 ± 0.10
4	0 (−)	1 (+)	0 (−)	0 (−)	0 (−)	0.2 (+)	0.2 (+)	0.02 (+)	0 (−)	0.1 (+)	WB + RS	7.66 ± 0.25	5.17 ± 0.09	31.42 ± 1.18
5	0 (−)	1 (+)	0.5 (+)	2 (+)	0 (−)	0.2 (+)	0.2 (+)	0 (−)	0.05 (+)	0 (−)	WB	8.73 ± 0.20	4.32 ± 0.16	451.44 ± 20.58
6	1 (+)	0 (−)	0.5 (+)	0 (−)	0 (−)	0 (−)	0.2 (+)	0.02 (+)	0.05 (+)	0 (−)	WB + RS	7.37 ± 0.24	5.17 ± 0.16	30.52 ± 1.01
7	1 (+)	1 (+)	0 (−)	2 (+)	0 (−)	0 (−)	0 (−)	0.02 (+)	0.05 (+)	0.1 (+)	WB	8.45 ± 0.10	4.37 ± 0.20	667.25 ± 28.08
8	0 (−)	0 (−)	0 (−)	2 (+)	0.5 (+)	0.2 (+)	0 (−)	0.02 (+)	0.05 (+)	0 (−)	WB + RS	6.93 ± 0.25	4.53 ± 0.20	18.67 ± 1.32
9	0 (−)	1 (+)	0.5 (+)	0 (−)	0.5 (+)	0 (−)	0 (−)	0 (−)	0.05 (+)	0.1 (+)	WB + RS	7.24 ± 0.07	5.27 ± 0.08	22.19 ± 2.01
10	0 (−)	0 (−)	0 (−)	0 (−)	0 (−)	0 (−)	0 (−)	0 (−)	0 (−)	0 (−)	WB	7.57 ± 0.46	5.31 ± 0.19	29.81 ± 1.41
11	1 (+)	0 (−)	0.5 (+)	2 (+)	0 (−)	0.2 (+)	0 (−)	0 (−)	0 (−)	0.1 (+)	WB + RS	8.63 ± 0.20	4.15 ± 0.07	353.25 ± 21.20
12	1 (+)	1 (+)	0.5 (+)	0 (−)	0.5 (+)	0.2 (+)	0 (−)	0.02 (+)	0 (−)	0 (−)	WB	7.69 ± 0.16	5.15 ± 0.04	29.09 ± 1.11

Note: Growth was measured in terms of log₁₀ CFU/mL. * Values are mean of three independent experiments with ± S.D. WB = wheat bran; WB + RS = wheat bran mixed with rice straw.

. These variables were peptone, meat extract, yeast extract, glucose, sodium acetate, di-potassium phosphate, triammonium citrate, magnesium sulphate, manganese sulphate, and solid substrate (wheat bran and wheat bran with rice straw). There was a total of 12 experiments, and each independent variable was represented at high (+) and low (−) levels. PBD was performed using the statistical software Design-Expert (version 6.0.7 Stat-Ease, Minneapolis, MN, USA). The response of each variable was determined using the following equation:

E_(xi) = 2(∑P_i⁺ − ∑P_i⁻)/N

where E_(xi) is the response of each variable. P_i+ and P_i− are the responses measured at high and low levels, respectively, and N is the number of experiments. An amount of 10 mL of each medium was prepared using the design and mixed with 5 g of wheat bran. All sets were inoculated with a culture of L. plantarum LD1 and monitored for growth and bacteriocin production. All experiments were performed in triplicate. The significant factors were investigated further for optimization [23].

2.4. Bioprocess Development Using Response Surface Methodology

Response surface methodology (RSM) using central composite design (CCD) was conducted to investigate the effects of the screened variables (yeast extract, peptone, glucose, and triammonium citrate) and the interaction between them. The CCD was performed using the statistical program Design-Expert (Stat-Ease, Minneapolis, MN, USA), as mentioned in

Table 3. Range of variables used for response surface methodology.

S. No.	Variables (%)	Actual Value of Each Coded Level
		−∞	−1	0	+1	+∞
1	Glucose	0.50	1.13	1.75	2.38	3.00
2	Peptone	0.25	0.69	1.13	1.56	2.00
3	Yeast Extract	0.25	0.69	1.13	1.56	2.00
4	Tri-ammonium citrate	0.10	0.20	0.30	0.40	0.50

. Each variable had five different levels of analysis (−∞, −1, 0, +1, and +∞), as mentioned in

Table 4. Growth, final pH, and bacteriocin production of Lactiplantibacillus plantarum LD1 in experimental design used in response surface methodology.

Run	Peptone (A)	Yeast Extract (B)	Glucose (C)	Tri-Ammonium Citrate (D)	Growth (log10 CFU/mL) *	Final pH *	Bacteriocin Production (AU/mL) *
1	+1	+1	−1	−1	8.80 ± 0.18	4.69 ± 0.22	502.40 ± 22.05
2	0	0	0	0	8.46 ± 0.40	4.51 ± 0.33	377.49 ± 18.29
3	0	0	0	0	8.29 ± 0.31	4.51 ± 0.33	377.49 ± 27.29
4	+1	+1	+1	+1	8.46 ± 0.34	4.54 ± 0.30	529.32 ± 19.05
5	+1	+1	+1	−1	8.41 ± 0.24	4.52 ± 0.33	307.48 ± 20.02
6	0	0	−∞	0	8.47 ± 0.38	4.68 ± 0.21	01.89 ± 0.12
7	0	0	0	0	8.29 ± 0.31	4.51 ± 0.33	329.46 ± 28.58
8	0	0	0	0	8.40 ± 0.38	4.51 ± 0.33	307.48 ± 23.20
9	−1	−1	+1	+1	8.23 ± 0.23	4.53 ± 0.30	241.38 ± 12.10
10	−1	−1	+1	−1	8.64 ± 0.20	4.52 ± 0.32	353.25 ± 24.15
11	+1	−1	+1	+1	8.86 ± 0.05	4.58 ± 0.25	241.38 ± 11.07
12	−1	−1	+1	−1	8.54 ± 0.09	4.54 ± 0.30	307.48 ± 22.13
13	+1	−1	−∞	−1	8.63 ± 0.18	4.62 ± 0.33	262.10 ± 25.02
14	0	0	−1	−∞	8.63 ± 0.11	4.61 ± 0.36	284.56 ± 28.01
15	+1	+1	0	+1	8.66 ± 0.22	4.61 ± 0.34	179.21 ± 17.02
16	−1	+1	0	+1	8.82 ± 0.07	4.57 ± 0.28	529.32 ± 31.06
17	−∞	0	−1	0	8.69 ± 0.19	4.63 ± 0.32	425.86 ± 38.01
18	+1	−1	−∞	−1	8.49 ± 0.25	4.37 ± 0.41	377.50 ± 21.05
19	+∞	0	−1	0	8.67 ± 0.19	4.59 ± 0.32	475.94 ± 27.02
20	0	0	−1	0	8.23 ± 0.23	4.36 ± 0.34	284.56 ± 20.12
21	−1	+1	−1	−1	8.72 ± 0.16	4.62 ± 0.33	425.86 ± 36.13
22	−1	−1	−1	+1	8.72 ± 0.24	4.50 ± 0.23	241.38 ± 12.10
23	0	+∞	0	0	8.58 ± 0.50	4.56 ± 0.29	502.40 ± 33.04
24	+1	−1	−1	+1	8.72 ± 0.24	4.59 ± 0.18	329.46 ± 19.17
25	−1	+1	+1	−1	8.85 ± 0.08	4.53 ± 0.24	451.44 ± 27.03
26	0	0	+∞	0	8.83 ± 0.06	4.59 ± 0.28	475.94 ± 36.01
27	0	−∞	0	0	8.37 ± 0.35	4.44 ± 0.28	158.96 ± 06.01
28	0	0	0	0	8.26 ± 0.27	4.35 ± 0.35	377.50 ± 18.06
29	0	0	0	+∞	8.56 ± 0.42	4.30 ± 0.45	582.86 ± 28.15
30	−1	+1	−1	+1	8.22 ± 0.20	4.47 ± 0.26	377.50 ± 07.26

* Values are mean of three independent experiments with ±S.D.

. There were in total 30 runs conducted using this design, with 24 axial points and six replicates at the center point. The growth and production of bacteriocin from L. plantarum LD1 was considered using a regression model, as suggested by Choi et al. [24].

2.5. Scale-Up of Growth and Bacteriocin Production

L. plantarum LD1 was grown in the optimized medium (peptone 1.13%, yeast extract 1.13%, glucose 1.56%, and tri-ammonium citrate 0.50%) containing different amounts (5–150 g) of wheat bran at 37 °C for 24 h. The growth (CFU/mL), pH reduction, and bacteriocin production (AU/mL) were monitored as described above.

2.6. Statistical Analysis

The responses of experimental and predicted values were evaluated using analysis of variance (ANOVA). The responses were assessed using a polynomial equation, and multiple regression analysis was performed to fit the data. Later, an experiment was carried out for evaluation of the responses, using the optimum conditions for variables determined via response surface methodology. All values are represented as mean with standard error.

3. Results and Discussion

Lactic acid bacteria (LAB) are known for their safety to humans and animals and suitability in industrial processes. They produce various antimicrobial compounds, including antibiotics, organic acids, acetoin, diacetyl, hydrogen peroxide, and bacteriocins. Bacteriocins, in particular, have gained attention as natural antimicrobials due to their inhibition of the growth of pathogens [11,12]. LAB have generally been grown in broth cultures for various applications. However, most of the conditions where these bacteria grow use natural solid substrates to deliver probiotic functions. Therefore, in this study, we used solid substrates for the growth of potential probiotic L. plantarum LD1 and production of bacteriocin in solid-state fermentation for commercial applications. These conditions will also provide an environmentally benign approach for the conversion of solid waste to produce value-added products [9].

3.1. Lignocellulosic Substrates for the Production of Bacteriocin in SSF

In wheat bran moistened with MRS medium, L. plantarum LD1 was able to grow to log₁₀ CFU/mL 8.15 ± 0.20 and produce 391.69 ± 0.58 AU/mL bacteriocin. On rice straw and rice bran, growth was log₁₀ CFU/mL 7.66 ± 0.05 and 8.09 ± 0.09 and bacteriocin production was 219.80 ± 0.58 and 380.73 ± 0.29 AU/mL, respectively. The growth and production of bacteriocin were log₁₀ CFU/mL 8.43 ± 0.13 and 284.56 ± 0.50 AU/mL, respectively, in the presence of sugarcane bagasse. The growth was recorded as log₁₀ CFU/mL 7.43 ± 0.05 and bacteriocin production 362.50 ± 0.05 AU/mL in mustard oil cake. The growth was also monitored on corn cobs, congress grass, oat bran, and chickpea bran and found to be log₁₀ CFU/mL 7.92 ± 0.03, 7.69 ± 0.37, 7.74 ± 0.24, and 6.16 ± 0.09, respectively, but the bacteriocin production was negligible (1.75 ± 0.23 to 1.80± 0.02 AU/mL). The pH was reduced after the growth from pH 6.0 to 4.64 [Table 1]. Thus, wheat bran, showing highest bacteriocin activity, was selected as the substrate for further optimization using MRS medium components in the RSM study. Similarly, different researchers have used various substrates such as bagasse, coffee husk, rice bran, ground nut oil cake, wheat bran, green gram flour, oats, corn flour, etc., for the growth of L. plantarum MTCC1407 [25]. In contrast, L. plantarum and Candida utilis were grown on a mixture of red jujube corn flour, peanut meal, and sodium hydrogen phosphate moistened with ultrapure water [3]. L. plantarum SN4 was grown on corn flour, soybean meal, peptide powder, and wheat bran moistened with basal medium [21]. In another study, saccharification of chestnut burrs, a lignocellulosic waste, was performed to produce bacteriocin using Lactobacillus plantarum CECT 211 [26].

3.2. Plackett–Burman Design

Plackett–Burman Design was used for identifying important independent variables affecting bacteriocin production. It was performed using eleven independent variables for growth and bacteriocin production by L. plantarum LD1 (Table 2). The growth in each set was found to be log₁₀ CFU/mL 6.31 ± 0.27 to 8.73 ± 0.20 and bacteriocin production was 1.89 ± 0.01 to 667.25 ± 0.08 AU/mL.

The effect of each independent variable is shown in a pareto graph (Figure 1). Yeast extract, beef extract, peptone, glucose, tri-ammonium citrate, magnesium sulphate, tween 80, and wheat bran were found to be significant factors. Out of these factors, glucose, peptone, yeast extract, and tri-ammonium citrate, which significantly influenced bacteriocin production, were selected for further study via RSM. Similarly, the optimum fermentation conditions for the growth of L. plantarum MTCC 1407 were evaluated using the sequential optimizing strategies PBD and RSM by Natarajan and Rajendran [25]. Plackett–Burman Design was also used to optimize the growth and lactic acid production of L. plantarum BL011, in which corn steep liquor, liquid acid protein residue of soybean, peptone, yeast extract, stirring speed, and aeration rate were used as independent variables. In this study, except corn liquor, all other variables were found to be significant for growth and lactic acid production [27]. There is no report available, to the best of our knowledge, for the growth of lactobacilli and production of bacteriocin on wheat bran. However, chestnut burrs and sugar cane bagasse have been used for the production of bacteriocin by different researchers [26,27,28]. Thus, the present study has additional value in the ongoing research on the cost-effective production of bacteriocins from L. plantarum LD1 under SSF conditions.

3.3. Response Surface Methodology

CCD was performed using four significant factors, i.e., peptone, yeast extract, tri-ammonium citrate, and glucose, achieved from PBD. The ranges of variables used in this study are given in Table 3. In total, 30 experiments were designed, with six replicates at the center point and 24 axial points. The response of each factor was analyzed by applying multiple regressions. The growth, final pH, and bacteriocin production recorded in each run are in Table 4. A high F value shows a low probability (p ˂ 0.0001). The model F-value of 87.26 implies the model is significant. Values of “Prob > F” less than 0.05 indicate that model terms are significant. In this case, A, B, C, D, A², B², C², D², AB, AC, AD, BC, BD, CD, B³, C³, D³, and BCD are significant model terms. The following quadratic regression function was obtained after analysis:

Y = −11.67 − 1.41A + 12.09B + 20.52C + 63.78D + 1.32A² − 4.35B² − 9.38β₃₃C² −
189.79D² + 1.19B³ + 1.60C³ + 225.00D³ − 0.91AB − 0.64AC + 1.71AD − 2.83BC −
21.94BD − 10.17CD + 11.89BCD.

Similarly, central composite design was performed to optimize the culture conditions of Pediococcus pentosaceus ET34 using sugarcane bagasse for the bacteriocin production. In this design, the temperature and hydrolysate of sugarcane bagasse (HSB) were taken as variables [28]. A statistical method based on RSM was performed to optimize the bacteriocin production by Lactobacillus acidophilus MS1. The bacteriocin activity was increased up to 2600 AU/mL using Box–Behnken experimental design [29]. Similarly, growth conditions of Lactobacillus plantarum BG24 were optimized for biomass production, where the R² value of the model was found to be 0.9931 and the F-value was 122.99, suggesting the fitness of the model [30].

The coefficient of determination (R²), which explains the fit of the model, was found to be 0.9930, suggesting the model terms are significant. Generally, a value of R² closer to 1 means that the model could predict a better response. Values greater than 0.1 indicate that the model terms are not significant. The results of interaction of different variables are mentioned in Figure 2a,b. Bacteriocin production varied with the concentrations of glucose, peptone, and yeast extract.

Bacteriocin production was found to be highest in run number 29, with 582.86 ±28.15 AU/mL. Run number 6 showed the lowest activity (1.89 ± 0.12 AU/mL). The rest of all the sets have bacteriocin activity in range of 158–475 AU/mL. The final pH in all sets was found to be in the range of pH 4.30–4.68. The variability in the response of bacteriocin production and consistency in expected and observed values suggests the model terms are fit (Table 3). Thus, wheat bran (5 g) moistened with 1.13% peptone, 1.13% yeast extract, 1.56% glucose, and 0.50% tri-ammonium citrate (run number 29) showing the highest activity was considered to be the optimal medium for the growth and production of bacteriocin by L. plantarum LD1. In another study, the concentration of yeast extract, ammonium sulphate, amount of substrate, and moisture content were optimized using fraction factorial design for lactic acid production from L. plantarum MTCC 6161 [31]. Response surface methodology and Box–Behnken experimental design were used to optimize the broth culture conditions for the production of antimicrobial compounds by Bacillus cereus [32].

3.4. Scale-Up of Growth and Bacteriocin Production in Optimized Condition

The growth of L. plantarum LD1, final pH, and bacteriocin production using different amounts of wheat bran viz. 5, 10, 50, 100, and 150 g are given in

Table 5. Growth (log₁₀ CFU/mL), final pH, and bacteriocin production (AU/mL) of Lactiplantibacillus plantarum LD1 in different culture volumes.

S. No.	Culture Volume (g)	Growth (log10 CFU/mL)	Final pH	Bacteriocin Activity (AU/mL)
1	5	8.56 ± 0.42	4.30 ± 0.45	582.86 ± 32.87
2	10	8.60 ± 0.38	4.31 ± 0.30	630.23 ± 23.76
3	50	8.63 ± 0.26	4.32 ± 0.23	601.13 ± 18.17
4	100	8.64 ± 0.05	4.32 ± 0.57	591.89 ± 33.04
5	150	8.64 ± 0.34	4.31 ± 0.34	592.04 ± 12.10

. The results showed sustainable production of bacteriocin using increasing amounts of wheat bran in SSF. The growth and final pH values are also similar in all the experiments. These results show that bacteriocin production under optimized conditions is sustainable and can be scaled up for large-scale production at the industrial level. Similarly, the production of pediocin PA-1 by Pediococcus pentosaceus ET34 was scaled up to a 2 L culture medium [28]. Thus, it was observed that growth and bacteriocin production were found to be similar when a higher culture volume was used. These findings support the successful scale-up of the bioprocess for the production of bacteriocin in SSF using wheat bran.

3.5. Cost-Effectiveness of the Bioprocess

The cost for the production of bacteriocin was 121,497.18 AU/USD in wheat bran moistened with MRS medium, which is 25% lower than the commercial MRS medium. Bacteriocin production was enhanced to 444,583.60 AU/USD in wheat bran moistened with the optimized medium. A low-cost culture medium was developed for bacteriocin Lac-B23 produced by L. plantarum J23 in submerged fermentation [33]. In another study, a modified MRS was developed for sakacin A production by L. sakei in liquid medium [34]. Thus, to the best of our knowledge, this is the first report to develop a low-cost medium for the growth of L. plantarum LD1 in SSF conditions.

4. Conclusions

The growth and bacteriocin production by Lactiplantibacillus plantarum LD1 in SSF conditions were optimized using statistical approaches. Bacteriocin production was almost 1.5-fold enhanced in the optimized medium, with a concomitant reduction in the cost of the medium. The production of bacteriocin was also scaled up using higher amounts of wheat bran, indicating the possibility of scaling up the optimized bioprocess. Thus, L. plantarum LD1 is able to grow on a solid substrate, wheat bran, and able to produce bacteriocin in an economical medium. These findings indicate that L. plantarum LD1 can be applied in solid foods and related clinical applications.