Sardasht black grape seeds were obtained from the Pakdis juice factory (Urmia, Iran). The kernels were dried for 2 h at 50°C to reach a moisture content of 6–7% and then stored in airtight packaging for further use. B. coagulans IBRC-M 10807 was obtained in freeze-dried form from the Iranian Biological Research Center (Tehran, Iran). Peptone from casein, glucose monohydrate, sodium chloride, yeast extract, magnesium chloride, calcium chloride, manganese chloride, Tween 80, hexane, chloroform, methanol, sodium sulfate, and isopropanol were purchased from Merck (Darmstadt, Germany). All chemicals are of analytical grade. Conjugated linoleic acid with 99% purity obtained from Sigma-Aldrich (St. Louis, Missouri, USA) was used as standard for determining CLA production.

Bacillus coagulans IBRC-M 10807 was grown for 37 h at 37°C, utilizing Nutrient Yeast Extract Salt Medium (NYSM) including glucose monohydrate (10 g/L), peptone from casein (5 g/L), sodium chloride (5 g/L), yeast extract (2–4%), magnesium chloride (0.2 g/L), calcium chloride (0.1 g/L), and manganese chloride (0.01 g/L), with 1% w/v Tween 80. After cultivation, the cultures were stored at 4°C and underwent three successive sub-culturing cycles within the same medium before each experiment.

The Soxhlet method and hexane solvent were used to extract oil from the dried kernels following Baydar et al. (2007). Briefly, dried seeds were ground in a grinder for 2 min, with 15-s intervals to prevent overheating the sample. Four grams of the powdered grape seeds were extracted using a Soxhlet extractor (Büchi Universal Extraction System B-811, Germany) for 6 h with 150 mL of hexane at 60°C.

Bacillus coagulans IBRC-M 10807 was cultured in NYSM broth under sterile conditions, and incubated for 24 h at 37°C. The culture medium was centrifuged at 5000 × g for 20 min and washed twice in 0.85% w/v sodium chloride solution. The final pellet containing the bacterial cells was added to sterile saline solution to reach a suspension with 10¹⁰ CFU/mL B. coagulans IBRC-M 10807 (Amiri et al., 2020).

NYSM broth was used to produce CLA by B. coagulans IBRC-M 10807. The pH of the culture medium was first adjusted using 1 N hydrochloric acid according to the statistical design, and the media was then sterilized at 121°C for 15 min. Next, grapeseed oil was added to the mixture with a syringe filter (pore size = 0.45 μm). Finally, 1 mL of the suspension containing 10¹⁰ CFU/mL of B. coagulans IBRC-M 10807 bacteria was added to 100 mL of NYSM broth and incubated according to the factorial experimental design order to determine the most effective factors of CLA production (Sekhavatizadeh et al., 2019; Amiri et al., 2021a).

For this purpose, five independent variables were evaluated: initial pH (5, 6 and 7), temperature (35, 40 and 45°C), incubation time (18, 33 and 48 h), grapeseed oil concentration (0, 500 and 1,000 μg/mL), and yeast extract concentration (0, 2 and 4%, w/w), as shown in Table 1. Analysis of variance was used to analyze the significance of the differences among variables at α ≥ 0.05. Numerical optimization was used to predict the optimal conditions based on the desirability function. The experimental design, statistical analysis, and numerical optimization were conducted with Design-Expert software version 12 (State-Ease Minneapolis, MN, USA).

Briefly, 10 mL of culture medium was centrifuged at 7,500 × g for 5 min at 4°C. Then, 6 mL of isopropanol (Merck, Darmstadt, Germany) was added to 3 mL of the supernatant and vortexed for 3 min. Next, 5 mL of hexane was added and vortexed for another 3 min. The mixture was centrifuged at 2,000 × g for 5 min at 4°C, and the total CLA was measured using a UV–Vis spectrophotometer (80–2088-64, Pharmacia LKB Biochrom, Cambridge, UK) at 233 nm against hexane as a blank. The CLA concentration was evaluated using a standard curve of conjugated linoleic acid at concentration of 0–30 mg/mL (Ribeiro et al., 2018).

Further characterization analyses were done on optimized sample to show the properties of bio produced CLA.

Accordingly, the results of this study also showed that initial pH, temperature, incubation time, as well as grapeseed oil and yeast extract concentrations had a significant effect on CLA production by B. coagulans IBRC-M 10807. As such, the total content of CLA was significantly increased by increasing all of the above factors (p < 0.05). The R² (coefficient of determination) and Adj-R² (adjusted coefficient of determination) values for Equation 1 were 0.977 and 0.959, respectively.

Where it is final equation in terms of coded factors: A is Initial pH, B is Temperature (°C), C is Incubation Time (h), D is Linoleic acid (%), and E is Yeast Extract (%).

Previous research has shown that fermentation conditions such as pH, temperature, time and the LA content of the medium affect the in vitro production of CLA (Terán et al., 2015; Hussain et al., 2016; Renes et al., 2017).

According to Figure 1A, the CLA production increased significantly by increasing the initial pH (p < 0.05). This finding is in line with the results of Özer et al. (2016), who showed that the CLA production by Lactobacillus plantarum at pH 6 was more than that produced at pH 5.5 and 5.7. Ye et al. (2013) indicated that near-neutral pH levels have a direct impact on cell growth and nutritional metabolism, which enhances the strain’s isomerizing capability, thereby increasing the conversion of linoleic acid to CLA. Previous research has highlighted that pH is a critical factor influencing the activity of the linoleate isomerase enzyme and the molecular synthesis of CLA, potentially serving as a limiting factor in CLA production (Dahiya and Puniya, 2018; Gorissen et al., 2010; Salsinha et al., 2018).

In addition to pH, temperature is also one of the most important parameters for enzymatic reactions. In this study, it was shown that increasing the temperature caused an increase in the amount of CLA (Figure 1B). This is in agreement with the research of He et al. (2015), who reported that an increase in the reaction temperature from 20°C to 37°C improved the yield of CLA. However, a further increase in reaction temperature led to a decline in CLA production, which aligns with findings from previous similar studies. These studies suggested that higher fermentation temperatures can reduce linoleate isomerase enzyme activity and inhibit bacterial growth (Gorissen et al., 2010; Kishino et al., 2010).

Incubation time had a significant effect on CLA yield (Figure 1C), as there was a positive correlation (p < 0.05) between incubation time and amount of CLA produced. Hosseini et al. (2015) also showed that CLA1 (cis-9, trans-11) production increased during incubation for up to 72 h, but then decreased after 96 h; the CLA2 (trans-10, cis-12) ratio increased with prolonged time. In addition, Özer and Kiliç (2020) reported that the amount of CLA increased by increasing fermentation time up to 73–79 h for L. plantarum, but similar to others’ findings, a further increase in the fermentation time caused a decrease in CLA production. This might be explained by the fact that high CLA concentration may have a toxic effect and thus inhibit further bacterial growth (Gorissen et al., 2015; Özer and Kiliç, 2020). Substrate concentration of the medium is another important factor that influences CLA yield. As shown in Figure 1D, the CLA production by B. coagulans IBRC-M 10807 increased by increasing the grapeseed oil concentration as the source of LA. This increase could be due to the use of grape seed oil. Although increasing free LA can have an antimicrobial effect, grape seed oil is not a completely pure precursor of free LA. So, it is a suitable precursor that does not have an antimicrobial effect by increasing the concentration to a certain extent. According to the literature, LA up to 1,000 μg/mL has no antimicrobial effect of probiotics (Amiri et al., 2020). On the other hand, grapeseed oil contains about 72–76% of LA, therefore, at the maximum concentration used, it is less than the antimicrobial limit of LA (Cao and Ito, 2003; Amiri et al., 2020). Similarly, Khosravi et al. (2015) found that CLA production significantly increased with the addition of 1–3 mg/mL of substrate to the medium. Wang et al. (2022) reported that the conversion rate of LA to CLA by L. acidophilus gently decreased as LA increased from 0.5 mg/mL to 2.0 mg/mL. In addition, Zhao et al. (2011) stated that the maximum amount of CLA (275.7 μg/mL) was obtained in the mixture containing 2 mg/mL linoleic acid; with the addition of more substrate, CLA production decreased. This may be related to the change in enzyme structure, which affects its function or because of antimicrobial effect of free LA in high concentrations. Yeast extract is rich in nutrients such as peptides and amino acids, therefore it is commonly used as a source of nitrogen to improve bacterial growth and metabolite production (Dahiya and Puniya, 2018). As shown in Figure 1E, the CLA yield increased with increasing concentration of yeast extract. Khosravi et al. (2015) also concluded that the total CLA formation increased at higher concentrations of yeast extract. Furthermore, similar results reported by Hennessy et al. (2009) indicated that the increase in yeast extract caused an increase in bacterial growth and CLA production.

In this study, the conditions of CLA production by B. coagulans were optimized using desirability function. The optimal conditions for CLA production were found to be initial pH of 6.8, temperature of 44.8°C, incubation time of 48 h, grapeseed oil concentration of 1,000 μg/mL, and yeast extract concentration of 3.88% with the desirability value equal to 1.00. Next, five samples were cultivated under these optimized conditions. There was no significant difference (p > 0.05) between actual amount (225.37 ± 0.74 μg/mL) and predicted values (219.48 μg/mL) of total CLA (Figure 2). The transformation efficiency of converting LA into CLA by B. coagulans IBRC-M 10807 was 33.78%. Iorizzo et al. (2024) reported that the transformation efficiency of converting LA into CLA by Lactiplantibacillus plantarum NCUL005 was 26.67%, and Salsinha et al. (2018) reported that the rate of conversion of LA to CLA ranged from 17.00 to 35.90% in MRS broth containing 200 μg/mL LA which are in agreement with the result of present study.

Gas chromatography analysis of total fatty acid methyl esters was used to identify the composition of CLA produced by B. coagulans IBRC-M10807. Two main CLA isomers were detected – CLA 1: c-9, t-11 C18:2, and CLA 2: t-10, c-12 C18:2 methyl esters. The main representative CLA isomers are 9c,11 t-18:2 and 10 t,12c-18:2. CLA has been shown to exercise diverse potent health effects such as antiobese, anticarcinogenic, antihypertensive and antidiabetic properties. The health effects of CLA depend on its isomers, for example the 10 t,12c isomer is anticarcinogenic, antiobese and antidiabetic, whereas the 9c,11 t isomer is mainly anticarcinogenic. These results are in accordance with several previous studies (Amiri et al., 2020; Razmjooei et al., 2020). As shown in Figure 3, the amount of CLA 1 is higher than CLA 2. According to prior research, CLA 1 has a stronger anti-inflammatory effect, however CLA 2 is known as an anti-obesity product (Razmjooei et al., 2020).

An organic molecule is a set of almost independent functional groups whose properties determine the physical and chemical properties of the compound, therefore the analysis of the functional group of organic compounds is important in identifying the compound. In order to determine the functional groups of CLA produced by B. coagulans, Fourier-transform infrared spectroscopy was used (Figure 4). The absorption at 3258.19 cm⁻¹ is due to a hydroxyl group (Costa et al., 2015), while the peak at 2934.53 cm⁻¹ indicates hydrocarbon chain asymmetric fatty acids (Amiri et al., 2020). The produced CLA has a sharp peak at 1896.94 cm⁻¹, which is related to carbonyl ester stretch (He et al., 2016), and the peak at 1084.91 cm⁻¹ indicates the tensile strength of a symmetric C–O ester (Salas-Valerio et al., 2022). The absorption at 1100 cm⁻¹ to 1,300 cm⁻¹ is related to the stretch of the C=O and C=C bonds, and the peak at 946.24 cm⁻¹ is a feature of the cis-trans conjugated bonds. Furthermore, the peak at 778.46 cm⁻¹ is due to methylene vibrations, which indicates high-chain fatty acids in CLA produced by B. coagulans (Amiri et al., 2020). Also, the peaks at 772.97 cm⁻¹ and 772.25 cm⁻¹ are detected in the generated CLA by different probiotic bacteria. These peaks correspond to methylene vibrations, and signify a characteristic feature of long-chain fatty acids, as described by Roach et al. (2002). The obtained results are consistent with the results of Amiri et al. (2020) and indicate the bio-production of CLA isomers with health-giving properties by probiotic B. coagulans IBRC-M 10807 from grape seed oil as a precursor.