Production of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 on a soybean agro-industrial waste-based medium: bioprocess optimization in STR bioreactors

Production of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 on a soybean agro-industrial waste-based medium: bioprocess optimization in STR bioreactors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Production of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 on a soybean agro-industrial waste-based medium: bioprocess optimization in STR bioreactors Dener Acosta de Assis, Marco A Záchia Ayub This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6596409/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Nov, 2025 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted 4 You are reading this latest preprint version Abstract The production and use of plant-based foods are increasing based on health, ethical, and environmental concerns. However, plants do not synthesize vitamin B 12 , thus the fortification of plant-based foods is highly recommended to prevent its deficiency. The aim of this work was to optimize vitamin B 12 production by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing on stirred tank bioreactors using the liquid acid protein residue of soybean, an agro-industrial waste, as medium culture. The influence of medium supplementation, pre-saccharification of sugars, and aeration strategies were investigated. The pH and temperature control were optimized applying the Design of Experiments (DoE) approach, using a central composite design. After the optimization process, the vitamin B 12 production more than tripled (from ~ 1.5 mg • L − 1 up to 5 mg • L − 1 ). Further, cultures produced high biomass concentrations for this bacterium (> 6 g • L − 1 ), whilst increasing by three-fold the specific biomass yield of vitamin B 12 (> 0.8 mg • g − 1 dry cell). This bioprocess based on cheap and available soybean agro-industrial waste and a GRAS microorganism can be an efficient alternative source of vitamin B 12 production to fortify plant-based products in the future. Sustainability agro-industrial residues plant-based foods cobalamin bioprocess technology Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Vitamin B 12 is an essential micronutrient required for human metabolism and blood cell synthesis. 1 Its deficiency leads to serious disorders, such as sensory and motor disturbances, depression, dementia, and, eventually, anemia and death. 2 The recommended human vitamin B 12 intake to prevent deficiency ranges from 4 to 20 µg • day − 1 for adults. 3 Peculiarly, humans rely almost entirely on animal-derived food (e.g. meat, fish, and dairy products) to obtain this micronutrient through conventional diets. 4 However, in the Anthropocene (the current geological age), industrial production of animal-based food has been proven to cause severe environmental impacts. 5 The sector occupies 75 of Earth’s arable land and it is the major driver for deforestation and biodiversity loss while responding to a massive greenhouse gas emission (> 9.7 billion t CO 2 -eq • year − 1 ), and 78 of all eutrophication of superficial waters worldwide. 6 – 11 According to the EAT Lancet Commission, a drastic shift toward more plant-based diets must happen in the near future to establish a truly sustainable food system and to avoid worsening scenarios of global warming and climate change. 12 In this sense, plant-based products are already growing in popularity. 13 , 14 In Europe, the sales of plant-based foods (e.g. meat, milk, and other plant-based analogues) increased by 49 % beween 2018 and 2020 (€ 2.4 billion vs. € 3.6 billion, respectively). 15 In the USA, the market share of the plant-based segment surpassed US $ 10 billion in 2022 and it is expected to double by 2029. 16 In this context, fortification of plant-based products with vitamin B 12 is crucial to prevent nutritional deficiencies, since plants do not synthesize or accumulate this essential micronutrient. 3 Vitamin B 12 is an expensive micronutrient. According to the ChemAnalyst, vitamin B 12 is worth around 1500 U $ per kg and its demand is expected to grow 5.45% in ten years 17 . Industrial production of vitamin B 12 is carried out exclusively through bioprocess due to its structural complexity. 18 The dairy Propionibacterium (dairy PAB) are particularly interesting bacterium because it holds the GRAS status and has the natural ability to synthesize vitamin B 12 in considerable amounts. 19 Recently, Propionibacterium freudenreichii subsp. shermanii ATCC 13673 has been reported to produce vitamin B 12 in an inexpensive and available soybean agro-industrial waste. 20 The Liquid Acid Protein Residue of Soybean (LAPRS) is an industrial effluent obtained during the soybean protein isolation process, and it is discharged into the environment as waste, after treatment. It possesses a high biochemical oxygen demand (> 20,000 mg O 2 • L − 1 ) due to its organic components (carbohydrates and soluble proteins), which turns it into a potential culture medium for bioprocess. In the study mentioned above, the LAPRS medium composition was optimized for vitamin B 12 biosynthesis by P. shermanii ATCC 13673 growing in agitating flasks. After the transfer of this bioprocess to bench STR bioreactors, appreciable amounts of vitamin B 12 were obtained (~ 2 mg • L − 1 ), however, the optimal growth condition was yet to be found. 20 Based on these considerations, the aim of this work was to optimize the vitamin B 12 production by P. shermanii ATCC 13673 growing in bench stirred tank bioreactors using the LAPRS as medium culture. Different bioprocess variables were investigated including aeration strategy (anaerobiosis followed by microaerophilic conditions or pure anaerobiosis) and pre-saccharification of complex soybean oligosaccharides. Additionally, temperature and pH control were optimized through design of experiments (DoE) approach. 2. Material and methods 2.1. Microorganisms and LAPRS medium culture The Propionibacterium freudenreichii subsp. shermanii ATCC 13673 strain was purchased from André Tosello Foundation (Campinas, SP, Brazil). The LAPRS was kindly donated by the International Flavors & Fragrances Inc. (Esteio, RS, Brazil) and collected and concentrated as described in 20 . The first LAPRS sample was used as substrate for preliminary experiments and the second and third LAPRS samples were used on the design of experiments (DoE) assays. 2.2. Chemical composition of LAPRS The LAPRS medium composition was previously described and optimized 20 . Therefore, the standard LAPRS medium culture for vitamin B 12 production by P. shermanii ATCC 13673 used in this work has the following composition: total sugars, 6–8 % protein 1 % cobalt, 250 mg • L − 1 ; Cysteine, 1,200 mg • L − 1 , and 5,6-Dimethilbenzimidazole (DMBI), 30 mg • L − 1 . Total carbohydrates, proteins, and ashes were measured for all LAPRS samples. The methodologies applied were Fehling test, Kjeldahl, and dry ashing, according to the Adolfo Lutz Institute protocols. 21 The pH was measured using a pH probe (PH0-14, KASVI). 2.3. Inoculum and growth in stirred tank bioreactors (STB) The inoculum and growth conditions were performed as previously described. 20 Briefly, cryopreserved P. shermanii ATCC 13673 cell suspensions were cultured at 30°C for 72 h to obtain the inoculum (optical density = 1 unit ABS 600 nm). The experiments were performed in 2 L stirred tank bioreactors (STB) Biostat® B Plus (Sartorius, Germany) operating with 1.5 L work volume, including 10 %(in volume) of inoculum. The LAPRS medium was sterilized in the cultivation vessel at 121°C, 15 min. Bioprocess standard controls were as follows: temperature, 30°C, pH 6.5 adjusted with NaOH 7 M and alternating aeration, in which the first 72 h of cultivation were carried out under anaerobiosis, followed by 96 h under microaerophilic conditions obtained by 0.5 vvm of air flow and 250 rpm using two Rushton turbines. 2.4. Preliminary experiments Preliminary experiments were performed to establish some bioprocess variables prior the optimization trough DoE approach. Additional supplementation, pre-saccharification, and the selection of aeration strategy were investigated in these experiments. Cobalt and DMBI are key components of vitamin B 12 structure. 18 In order to check whether their concentration would impact vitamin B 12 production in bioreactors, we carried out an experiment increasing their concentration by 20 %in relation to the standard medium described in the section 2.2. The carbohydrates present in the LAPRS are mostly complex soybean oligosaccharides. 20 Therefore, a pre-saccharification step was also investigated. For this reaction, based on Coghetto et al., 22 a combination of invertase (1,000 U • L − 1 ) and alpha-galactosidase (1,800 U • L − 1 ) were used at 50°C, pH 4.5 for 3 h, reaching a hydrolysis degree of ~ 60 %. wo different aeration strategies are often applied for dairy PAB growth and vitamin B 12 production (i.e. anaerobiosis/microaerophilic conditions or pure anaerobiosis). Here, we compared them under the following conditions: 1) alternating anaerobiosis (72 h) followed by microaerophilic conditions (96 h); and 2) culture run exclusively under anaerobiosis (168 h). The experiment performed under standard conditions, as described in section 2.3., was coded as LA30. The experiments with additional supplementation, pre-saccharification, and exclusively anaerobiosis were coded as: LA30+, HA30, and HAN30, respectively. 2.5. Central composite design 2 2 After the initial tests, a central composite design 2 2 was applied to find the optimal temperature and pH values for vitamin B 12 production by P. shermanii ATCC 13673 growing in the LAPRS based medium. The variables levels tested in this study are shown in Table 1 . The vitamin B 12 specific yield was chosen as response variable for statistical analysis. Table 1 Temperature and pH levels studied in the central composite design 2 2 to optimize vitamin B 12 production by P. shermanii in STB. Variables Level − 1.41 − 1 0 + 1 + 1.41 pH 5.08 5.5 6.5 7.5 7.91 Temperature 28.5 30 33.5 37 38.5 2.6. Biomass, vitamin B 12 , monosaccharides, and organic acids analyses Biomass was measured by the gravimetric method and the results were expressed as dry weight of cells. Organic acids were analyzed using HPLC-RID system equipped with Bio-Rad HPX-87H column and H 2 SO 4 5 mM solution as mobile phase. Monosaccharides (glucose, galactose, and fructose) were measured by HPLC system equipped with Bio-Rad HPX-87C column using Milli-Q water as mobile phase. Vitamin B 12 analysis was performed with a validated method previously described. 20 Briefly, 20 mL of broth were centrifuged, the cellular pellet was washed with distilled water and resuspended in 20 mL of extraction buffer (phosphate 0.1 M; KCN, 0.1 %weight fraction; pH 4.5). The extraction was performed in amber flasks, using autoclave (Phoenix, Brazil) at 121°C for 15 min. The extracts were centrifuged, and the supernatant was collected, filtered through acetate cellulose membranes (0.22 µm) and frozen (-18°C) until HPLC-DAD analysis using C18 column Shimadzu and a gradient elution of Milli-Q water:methanol. 2.7. Statistical analysis Statistical analyses (ANOVA and Tukey’s test) were performed using the GraphPad Prism version 9.5.1 (GraphPad software, USA). The mathematical modelling of the central composite design 2 2 was performed using the software Statistica (v. 10.0, StatSoft, USA). 3. Results and Discussion 3.1. LAPRS chemical composition The LAPRS is a nutrient-rich agro-industrial residue. The chemical composition of the LAPRS samples used as culture medium in this work is shown in the Table 2 . Carbohydrates are the major component in the LAPRS (up to 8% m/v). Most of them are presented as sucrose, stachyose, raffinose, the main sugars of soybean by-products. 20 Therefore, the enzymatic pre-treatment using hydrolytic enzymes may be necessary to liberate the monosaccharides and improve the bioprocess and it was investigated in this study. Table 2 Chemical composition of the LAPRS samples used as the culture medium for vitamin B 12 production. Analyses Results LAPRS-industrial sample 1 LAPRS-industrial sample 2 LAPRS-industrial sample 3 pH 4.60 ± 0.01 a 4.52 ± 0.02 ab 4.43 ± 0.01 b Carbohydrates (%) 6.21 ± 0.19 b 6.84 ± 0.01 b 8.38 ± 0.10 a Proteins (%) 1.27 ± 0.02 b 1.71 ± 0.06 a 1.43 ± 0.02 b Ashes (%) 1.67 ± 0.17 a 1.67 ± 0.25 a 1.63 ± 0.08 a The means with the same letters in the rows indicates no statistical difference (Tukey’s test, p-value < 0.05). Proteins are present in lower amounts than sugars (1–2% m/v) in the LAPRS (Table 2 ), but in adequate concentrations to enable microbial growth, without any nitrogen supplementation. This fact has been shown in a previous work, where the supplementation of nitrogen did not produce any effect on vitamin B 12 specific yields by P. shermanii in batch fermentations using LAPRS-based medium culture. 20 On the other hand, it is important to note that the specific supplementation of one aminoacid, cysteine, is necessary. Cysteine is involved in S-Adenosyl methionine (SAM) regeneration, which is an important co-factor for vitamin B 12 formation. 23 The supplementation of LAPRS culture medium with cysteine proved to be positive for vitamin B 12 accumulation by the P. shermanii ATCC 13673, hence, it was kept in this work. 20 The LAPRS shows a mineral content of up to 1.6% m/v (Table 2 ). It includes minerals required for microbial development, such as potassium (K), phosphorus (P), manganese (Mn), and magnesium (Mg) 20 . However, cobalt (Co) is not present in the LAPRS, and its supplementation is required because it is a central component of the vitamin B 12 molecule structure. The acid pH (pH 4.5) is a characteristic of the LAPRS due to the protein precipitation at the isoelectric point. 22 Nonetheless, the pH was controlled automatically in all fermentations according to the experimental design. It is worth to mention that a significant variation (ANOVA, p < 0.05) was observed within pH, carbohydrate and protein content within the LAPRS industrial samples (Table 2 ). This was expected since LAPRS is an agro-industrial by-product and its composition is vastly influenced by the vegetable matrix and the manufacturing practices. However, the observed variation did not affect the strain growth kinetics (e.g. biomass production or substrate consumption), which highlights the robustness of our bioprocess. 3.2. Preliminary experiments Figure 1 depicts the growth kinetics of P. shermanii ATCC 13673 in the standard LAPRS medium and in the same medium with 20% increase of vitamin B 12 precursors (i.e. cobalt and DMBI). The growth patterns of P. shermanii ATCC 13673 were similar independently of the supplementation strategy, and there was no statistical difference between treatments on bioprocess yields (Table 3 ). The only exception it was the propionate titer that decreased from 9.61 g • L − 1 to 6.77 g • L − 1 between LA30 and LA30+, respectively. It had statistical significance (ANOVA, p < 0.05), but did not impact the other yields. Table 3 Results obtained on the optimization of vitamin B 12 production by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using LAPRS based medium on STB. Experiment Biomass (g • L − 1 ) Substrate consumption (g • L − 1 ) Propionate (g • L − 1 ) Acetate (g • L − 1 ) Vitamin B 12 (mg • L − 1 ) Yields B 12 (mg • g − 1 biomass) Q B12 mg • (L • h) −1 LA30 6.23 ± 0.23 ab 10.41 ± 0.57 b 9.61 ± 0.74 a 8.76 ± 1.22 ab 1.46 ± 0.37 b 0.24 ± 0.07 b 0.01 ± 0.002 b LA30 + 7.77 ± 1.31 ab 13.22 ± 2.85 b 6.77 ± 0.57 b 5.12 ± 2.10 bc 1.89 ± 0.48 b 0.24 ± 0.03 b 0.01 ± 0.004 b HA30 8.47 ± 0. 09 a 21.80 ± 0.61 a 7.56 ± 0.06 ab 10.21 ± 0.53 a 4.16 ± 1.03 ab 0.50 ± 0.12 ab 0.02 ± 0.001 ab HAN30 5.35 ± 0.42 b 22.22 ± 0.30 a 9.59 ± 0.88 a 3.04 ± 0.28 c 4.00 ± 0.46 ab 0.71 ± 0.12 a 0.02 ± 0.001 ab VAL32 6.72 ± 0.02 ab 22.24 ± 1.29 a 8.37 ± 0.15 ab 3.65 ± 0.38 c 5.64 ± 0.09 a 0.84 ± 0.01 a 0.03 ± 0.001 a All results correspond to the culture time of 168 h, except for propionate values that are related to samples taken at 72 h of growth. The means with the same letters in columns indicates no statistical difference (Tukey’s test, p-value < 0.05). LA30: Experiment performed under standard conditions; LA30 +: Experiment performed with additional supplementation (20% increase of vitamin B 12 precursors); HA30: Experiment performed with hydrolyzed LAPRS and standard supplementation; HAN30: Experiment performed with hydrolyzed LAPRS, standard supplementation under anaerobiosis; VAL32: Experiment performed with hydrolyzed LAPRS, standard supplementation under anaerobiosis and optimized pH (6.5) and temperature (32°C) control. Previously, it was noticed that the transfer of this bioprocess using LAPRS-based medium from shaking flasks to bench STB increased the biomass yield of P. shermanii ATCC 13673 up to 4 folds. On the other hand, the vitamin B 12 specific yield (Y B12 ) decreased from 0.6 mg • g − 1 to 0.25 mg • g − 1 . 20 In this context, we hypothesized that essential vitamin B 12 precursors could be lacking. However, as shown in Table 3 , the additional supplementation did not increase the vitamin B 12 specific yield, which remained 0.24 mg • g − 1 . In this case, the selective increase in biomass yield can be attributed just to the better variables control in STB (the pH and alternation aeration) and not the lack of vitamin B 12 precursors. Therefore, the standard medium was kept in further experiments. The availability of easily fermentable sugars is another key factor in bioprocess. Since LAPRS based medium is rich in complex carbohydrates (stachyose and raffinose), the influence of a pre-saccharification process was also investigated. Figure 2 A shows the growth kinetics of P. shermanii ATCC 13673 on the hydrolyzed LAPRS medium. The hydrolysis at least doubled the substrate consumption (21.8 g • L − 1 ), vitamin B 12 titer (4.16 mg • L − 1 ) and specific yield of B 12 (0.5 mg • g − 1 ), compared to LA30 experiment (Table 3 ). Unlike the other yields, propionate and acetate did not have significant changes between LA30 and HA30 experiments. This feature of the P. shermanii ATCC 13673 is desirable for vitamin B 12 production since high amounts of propionate (> 10 g • L − 1 ) may slow down the cell growth or even stop it due to end-product inhibition. 24 Monosaccharides usually enables microbes a faster growth and metabolic activity due to less energy required for the substrate breakdown. 25 The higher metabolic flow produces more energy and the synthesis of vitamin B 12 precursors, thus increasing its yield. 23 Glucose and galactose were the sugars metabolized by P. shermanii ATCC 13673, with negligible fructose consumption (Table S1 ). The preference for glucose rather than fructose by dairy PAB has been reported by Piwowarek et al., 25 who show that P. freudenreichii T82 was able to completely deplete glucose as substrate, but only half the amount of fructose under the same conditions and time. Based on our findings, the glucose and galactose were considered the total sugars in the afterwards experiments using the hydrolyzed LAPRS-based medium. Different aeration strategies (alternating anaerobiosis/microaerophilic conditions and pure anaerobiosis) have been tested for vitamin B 12 production by Propionibacterium genus. To decide the best condition both strategies were evaluated for P. shermanii ATCC13673 growth using the hydrolyzed LAPRS medium culture. Figure 2 A shows the strain growth under alternating aeration (experiment HA30), while Fig. 2 B shows the results for anaerobiosis (experiment HAN30). The aeration after 72 h had significant effect on biomass and acetate accumulation compared to the culture under anaerobiosis (Table 3 ). This phenomenon is typical of alternating aeration strategy. The oxygen triggers conversion of propionate to acetate and increases energy production by the strain, which leads to higher biomass and acetate production, as observed in the HA30 experiment. 26 However, the alternating aeration strategy did not influence the volumetric production of vitamin B 12 compared to the exclusively anaerobic culture (4.16 mg • L − 1 and 4.00 mg • L − 1 , respectively). In fact, the anaerobiosis enabled P. shermanii ATCC 13673 to reach a higher specific yield of vitamin B 12 compared to the alternating aeration strategy (0.71 mg • g − 1 and 0.50 mg • g − 1 , respectively). Microbial biosynthesis of vitamin B 12 follows two distinct routes, known as: aerobic and anaerobic routes. 18 Dairy PAB follows the anaerobic route and even low volumetric oxygen transference coefficient (K L a < 10 h − 1 ) at the early growth phase significantly affects the vitamin B 12 biosynthesis. 27 On the other hand, these bacteria use oxygen at late growth phase for DMBI synthesis (essential to produce active vitamin B 12 ) via Blub/CobT2 enzyme. 28 Additionally, aeration triggers propionate consumption and can attenuate end-product growth inhibition. 26 , 29 , 30 Hence, alternating aeration along the culture have been widely used in bioprocess for vitamin B 12 production with dairy PAB. 31 , 32 , 33 However, when DMBI is supplemented to the culture medium, it seems that the oxygen-dependent DMBI synthesis do not contribute significantly for vitamin B 12 production. Our findings suggest that under anaerobiosis, secured the external DMBI supplementation, dairy PAB can accumulate significantly more vitamin B 12 per gram of cells (Table 3 ). Therefore, anaerobiosis was chosen for vitamin B 12 production in the following experiments. Temperature and pH are also important variables to investigate during the bioprocess development. They affect dairy PAB growth rate and vitamin B12 production. Different temperatures (28°C to 38°C) and pH (5 to 7) values have been considered for dairy PAB cultures. 34 , 35 , 36 , 37 Therefore, after the testing the previous bioprocess variables, the optimum values of these two parameters were investigated using the DoE approach. 3.3. Bioprocess optimization by Design of Experiments (DoE) A central composite design 2 2 was applied for the optimization of temperature and pH values. Ten independent experiments were performed according to the experimental design presented in Table 4 , including two replicates at the central point to estimate the error. Table 4 Experimental matrix of the central composite design 2 2 with two duplicates at central point and the vitamin B 12 specific yield produced by P. shermanii ATCC 13673 growing in STB. Experiment Independent variables Response variable pH Temperature (°C) Yields B 12 (mg • g − 1 dry cell) 1 5.5 30 0.75 2 7.5 30 0.42 3 5.5 37 0.26 4 7.5 37 0.17 5 6.5 28.5 0.41 6 6.5 38.5 0.16 7 5.08 33.5 0.35 8 7.91 33.5 0.25 9 6.5 33.5 0.69 10 6.5 33.5 0.76 The mathematical modeling of the results based on the experimental data was statistically significant (ANOVA, p-value < 0.05) and presented a satisfactory coefficient of determination (R 2 = 0.89). The equation (Eq. 1) explained ~ 90% of the vitamin B 12 biosynthesis by P. shermanii ATCC 13673 growing in STB. Eq. 1 YB 12 = -19.2161 + 1.7898A – 0.1873A 2 + 0.8959B – 0.0156B 2 + 0.0171AB The response surface obtained through the mathematical modeling is illustrated in Fig. 3 . Both variables presented a negative effect on vitamin B 12 production by P. shermanii ATCC 13673 (Figure S1 ). Thus, maximum vitamin B 12 specific yields were observed around the lowest to the central levels tested, respectively pH 5.5 to 6.5, and temperature 30°C to 33.5°C. This finding is in accordance with previous reports for dairy PAB growth and propionate production as well. 37 – 39 The mathematical modelling predicted optimal vitamin B 12 specific yields (> 0.76 mg • g − 1 ) controlling pH and temperature at 6.2 and 32°C, respectively. The specific yields of vitamin B 12 obtained experimentally was 0.84 mg • g − 1 , validating the mathematical prediction. 3.4. Growth kinetics of P. shermanii ATCC 13673 under optimized conditions Figure 4 illustrates the growth kinetics of P. shermanii ATCC 13673 under optimized conditions (experiment VAL32). The peak of biomass and vitamin B 12 production were obtained at 72 h and 96 h of culture, respectively. It represents a significant increase in productivity compared to the first experiment LA30. In addition, the vitamin B 12 specific yields more than tripled compared to the same experiment (Table 3 ). On the other hand, the biomass production and vitamin B 12 specific yield did not differ statistically from the previous experiment HAN30 (Table 3 ). Yet, an increase trend was observed in both outcomes. Biomass went from 5.35 g • L − 1 to 6.72 g • L − 1 , respectively, and vitamin B 12 specific yield from 0.71 mg • g − 1 to 0.84 mg • g − 1 , respectively. Additionally, the vitamin B 12 titer in the VAL32 experiment it was the highest of them all (5.64 mg • L − 1 ). This indicates that the optimization of temperature and pH values improved P. shermanii ATCC 13673 biomass production, at the same time increasing the vitamin B 12 synthesis. Vitamin B 12 production by dairy PAB fluctuates according to the strain, medium culture composition and other bioprocess variables, such as the culture technique applied (batch, perfusion bioreactor, extractive fermentation, among others). 40 Under the conditions of the batch process, using synthetic medium, vitamin B 12 titer produced by these microorganisms usually ranges from 6 to 30 mg • L − 1 with specific yields around 0.6 to 0.9 mg • g − 1 of dry cells. 19 , 23 , 30 , 41 , 42 . Using agricultural wastes as substrate, the outcomes of vitamin B 12 can be less expressive. Hedayti et al., 36 obtained approximately 3 mg • L − 1 of vitamin B 12 by Propionibacterium freudenreichii PTCC1674 growing in rice bran substrate. Similar results were reported by Hajfarajollah et al., 43 (2.74 mg • L − 1 ) in a bioprocess using Propionibacterium freudenreichii PTCC1674 and sunflower waste oil. In this work, after the optimization process, a considerable amount of vitamin B 12 was obtained (5.64 mg • L − 1 ) using the LAPRS medium culture. Moreover, the specific yields reached (0.84 mg • g − 1 dry cells) were as high as those reported for dairy PAB growing in synthetic media. This bioprocess based on agro-industrial waste and a GRAS microorganism can be an alternative to be used in the fortification of plant-based foods. The bioaccessibility of vitamin B 12 delivered by dairy PAB biomass has been proven to be as high as the pharmaceutical form of vitamin B 12 . 44 Commercial vitamin B 12 production uses refined substrates, non-food grade strains (i.e. Pseudomonas denitrificans) and requires expensive downstream processing to obtain the highly purified pharmaceutical vitamin B 12 31 . On the other hand, in this work was utilized a cheap and highly available soybean agro-industrial waste as substrate. Additionally, P. shermanii ATCC 13673 is a GRAS microorganism whose biomass could be added directly to plant-based food products to fortify them, without the expensive downstream steps (e.g. cell lysis, crystallization etc .). This unique combination, coupled to the high vitamin B 12 yields obtained after optimization, may reduce costs for vitamin B 12 fortification of plant-based foods and highlights the importance of the bioprocess developed in this study. 4. Conclusion The vitamin B 12 production of P. shermanii ATCC 13673 grown in STB using LAPRS as medium culture was successfully optimized, being a new, cheap, and safe alternative to produce this important vitamin using exclusively plant-based medium. The enzymatic hydrolysis of complex carbohydrates present in LAPRS enabled the strain to produce considerable amounts of vitamin B 12 . Concerning aeration, anaerobiosis was the most suitable strategy for vitamin B 12 production by P. shermanii ATCC 13673. Our results suggest that, when DMBI supplementation is secured, an alternating aeration strategy may not be required for active vitamin B 12 synthesis by dairy PAB. Finally, DoE approach allowed us to find the optimal temperature, and pH control was established for efficient biomass production (6.72 g • L − 1 ) and vitamin B 12 yields (5.64 mg • L − 1 and 0.84 mg • g − 1 dry cell) by P. shermanii ATCC 13673, whose biomass could be used to fortify plant-based products in the food industry. Declarations Work’s Funding This work was funded by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq, Brazil), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, RS), through grants from Project RITES TO 22/2551-0000397-4, and scholarships from CAPES Code Fund 001 (Brazil). Authors’ Contributions D. A. 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Biotechnol Prog Published online 2020:1–9. 10.1002/btpr.3011 Instituto Adolfo Lutz. (2008). Métodos Físico-Químicos Para Análise de Alimentos (4th ed.). Instituto Adolfo Lutz. Coghetto, C. C., Vasconcelos, C. B., Brinques, G. B., & Ayub, M. A. Z. (2016). Lactobacillus plantarum BL011 cultivation in industrial isolated soybean protein acid residue. Brazilian Journal of Microbiology , 47 , 941–948. 10.1016/j.bjm.2016.06.003 Liu, J., Liu, Y., Wu, J., Fang, H., Jin, Z., & Zhang, D. (2021). Metabolic profiling analysis of the vitamin B12 producer Propionibacterium freudenreichii. Microbiologyopen , 10 (3), 1–19. 10.1002/mbo3.1199 Wang, P., Wang, Y., & Su, Z. (2012). Microbial production of propionic acid with Propionibacterium freudenreichii using an anion exchanger-based in situ product recovery (ISPR) process with direct and indirect contact of cells. 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World Journal Of Microbiology & Biotechnology , 14 , 1996–1999. https://doi.org/10.1023/A:1008868907251 Deptula, P., Kylli, P., Chamlagain, B., Holm, L., Kostiainen, R., & Piironen, V. (2015). BluB / CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B 12 by Propionibacterium freudenreichii. Microbial Cell Factories , 14 , 1–12. 10.1186/s12934-015-0363-9 Furuichi, K., Hojo, K., ichi, Katakura, Y., Ninomiya, K., & Shioya, S. (2006). Aerobic Culture of Propionibacterium freudenreichii ET-3 Can Increase Production Ratio of 1,4-Dihydroxy- 2-Naphthoic Acid to Menaquinone. J Biosci (Rajshari) , 101 , 464–470. 10.1263/jbb.101.464 Miyano, K., Ye, K., & Shimizu, K. (2000). Improvement of vitamin B12 fermentation by reducing the inhibitory metabolites by cell recycle system and a mixed culture. Biochemical Engineering Journal , 6 , 207–214. 10.1016/S1369-703X(00)00089-9 Calvillo, Á., Pellicer, T., Carnicer, M., & Planas, A. (2022). Bioprocess Strategies for Vitamin B12 Production by Microbial Fermentation and Its Market Applications. Bioengineering , 9 (8), 1–24. 10.3390/bioengineering9080365 Chamlagain, B., Deptula, P., Edelmann, M., et al. (2016). Effect of the lower ligand precursors on vitamin B12 production by food-grade Propionibacteria. LWT - Food Science and Technology , 72 , 117–124. 10.1016/j.lwt.2016.04.023 Xie, C., Coda, R., Chamlagain, B., et al. (2021). Fermentation of cereal, pseudo-cereal and legume materials with Propionibacterium freudenreichii and Levilactobacillus brevis for vitamin B12 fortification. LWT , 137 , 110431. 10.1016/j.lwt.2020.110431 Coral, J., Karp, S. G., Vandenberghe, L. P., de Parada, S., Pandey, J. L., & Soccol, A. (2008). Batch Fermentation Model of Propionic Acid Production by Propionibacterium acidipropionici in Different Carbon Sources. Applied Biochemistry And Biotechnology , 151 , 333–341. 10.1007/s12010-008-8196-1 Feng, X., Xu, H., Yao, J., Li, S., Zhu, H., & Ouyang, P. (2010). Kinetic Analysis and pH-Shift Control Strategy for Propionic Acid Production with Propionibacterium. Applied Biochemistry And Biotechnology , 160 , 343–349. 10.1007/s12010-008-8300-6 Hedayati, R., Hosseini, M., & Najafpour, G. D. (2020). Optimization of semi-anaerobic vitamin B12 (cyanocobalamin) production from rice bran oil using Propionibacterium freudenreichii PTCC1674. Biocatalysis And Agricultural Biotechnology , 23 , 101444. https://doi.org/10.1016/j.bcab.2019.101444 Seshadri, N., & Mukhopadhyay, S. N. (1993). Influence of environmental parameters on propionic acid upstream bioprocessing by Propionibacterium acidi-propionici. Journal Of Biotechnology , 29 , 321–328. https://doi.org/10.1016/0168-1656(93)90063-S Piwowarek, K., Lipińska, E., Hać-szymańczuk, E., Rudziak, A., & Kieliszek, M. (2019). Optimization of propionic acid production in apple pomace extract with Propionibacterium freudenreichii. Preparative Biochemistry & Biotechnology , 49 , 974–986. 10.1080/10826068.2019.1650376 Zhuge, X., Liu, L., Shin, H., Li, J., Du, G., & Chen, J. (2014). Improved propionic acid production from glycerol with metabolically engineered Propionibacterium jensenii by integrating fed-batch culture with a pH-shift control strategy. Bioresource Technology , 152 , 519–525. 10.1016/j.biortech.2013.11.063 Assis, D. A., Machado, C., Matte, C., & Ayub, M. A. Z. (2022). High Cell Density Culture of Dairy Propionibacterium sp. and Acidipropionibacterium sp.: A Review for Food Industry Applications. Food Bioproc Tech , 15 (4), 734–749. 10.1007/s11947-021-02748-2 Wang, P., Wang, Y., & Su, Z. Improvement of Adenosylcobalamin Production by Metabolic Control Strategy in Propionibacterium freudenreichii. Published online 2012:62–72. 10.1007/s12010-012-9654-3 Wang, P., Jiao, Y., & Liu, S. (2014). Novel fermentation process strengthening strategy for production of propionic acid and vitamin B12 by Propionibacterium freudenreichii. Journal Of Industrial Microbiology And Biotechnology , 41 , 1811–1815. 10.1007/s10295-014-1513-5 Hajfarajollah, H., Mokhtarani, B., Mortaheb, H., & Afaghi, A. (2015). Vitamin B12 biosynthesis over waste frying sunflower oil as a cost effective and renewable substrate. Journal Of Food Science And Technology , 52 , 3273–3282. 10.1007/s13197-014-1383-x Chamlagain, B., Peltonen, L., Edelmann, M., et al. (2021). Bioaccessibility of vitamin B12 synthesized by Propionibacterium freudenreichii and from products made with fermented wheat bran extract. Curr Res Food Sci , 4 , 499–502. 10.1016/j.crfs.2021.07.009 Supplementary Files OptmB12SM.docx Cite Share Download PDF Status: Published Journal Publication published 15 Nov, 2025 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted Reviewers agreed at journal 13 May, 2025 Reviewers invited by journal 13 May, 2025 Editor invited by journal 08 May, 2025 First submitted to journal 05 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6596409","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455837367,"identity":"eda3f3c5-6a57-47a4-916c-032658c62702","order_by":0,"name":"Dener Acosta de Assis","email":"","orcid":"","institution":"UFRGS: Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Dener","middleName":"Acosta","lastName":"de Assis","suffix":""},{"id":455837368,"identity":"ffbef775-d824-4906-9ba2-9a798616736b","order_by":1,"name":"Marco A Záchia Ayub","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYPCCA3IMzAfADOK1GDOwJZCoJbGBaC267ccffuZtu5O+4Rjv45c/GO7kE9RidiYhWZq37VnuhmPsZtY8DM8sGwhqOZBwAKjlcO6G+21sxgwMhw0I23L+YfNvoJZ0g2NsbIY/iNJyI5kNZEsCUAvzAx7itDxjs5xz7pnhTKAtzDwGz4hxWPrjG2/K7sjzAW35+KPiDmEtIMDEA6HZJBiI08DAwPgDQjN/IFLDKBgFo2AUjDAAAKpXQnywv31zAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4410-6336","institution":"UFRGS: Universidade Federal do Rio Grande do Sul","correspondingAuthor":true,"prefix":"","firstName":"Marco","middleName":"A Záchia","lastName":"Ayub","suffix":""}],"badges":[],"createdAt":"2025-05-05 17:21:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6596409/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6596409/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12010-025-05402-1","type":"published","date":"2025-11-15T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82809724,"identity":"dde161d9-4eb2-49d3-a80d-a7c83c527c18","added_by":"auto","created_at":"2025-05-15 13:13:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1223852,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth kinetics of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using standard LAPRS medium (LA30, graph A), and the same medium with 20 % increase in vitamin B12 precursors (LA30 +, graph B). Biomass (▪), total sugars (●), propionic acid (△), acetic acid (○), and vitamin B12 (•). The bioprocess variables were controlled a 30 °C, pH: 6,5, stirring: 250 rpm, under anaerobiosis from 0 h to 72 h, followed by aeration (air flow 0.5 vvm) until the culture end (168 h). DMBI was supplemented after 72 h of fermentation in both experiments. Results represent the mean of duplicates.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6596409/v1/52da4214a2ec91968ed2a8bb.png"},{"id":82809743,"identity":"bfc5313c-7cb9-4802-90a9-c598381b247f","added_by":"auto","created_at":"2025-05-15 13:13:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1220136,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth kinetics of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using standard LAPRS medium culture pre-treated with hydrolytic enzymes under anaerobic/microaerophilic conditions (HA30, graph A) and anaerobiosis (HAN30, graph B). Biomass (▪), total sugars (●), propionic acid (△), acetic acid (○), and vitamin B12 (▫). The bioprocess conditions were controlled as 30 °C, pH: 6,5, and stirring: 250 rpm. DMBI was supplemented after 72 h of fermentation in both experiments. Results represent the mean of duplicates.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6596409/v1/d5ee6729390a60086f1e662f.png"},{"id":82809725,"identity":"fe9a52aa-117f-4186-951e-c2cf5f7733f2","added_by":"auto","created_at":"2025-05-15 13:13:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29813,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface obtained in the optimization of temperature and pH variables through central composite design 2\u003csup\u003e2 \u003c/sup\u003eusing specific yields of vitamin B\u003csub\u003e12\u003c/sub\u003e as response variable.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6596409/v1/64a065ea5fe5eb49031e91cc.png"},{"id":82809727,"identity":"07a16608-8ca8-4eaa-be59-5acaaf348f56","added_by":"auto","created_at":"2025-05-15 13:13:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1035823,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth kinetics of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 under optimized conditions (VAL32). Biomass (▪), total sugars (●), propionic acid (△), acetic acid (○), and vitamin B12 (•). The bioprocess was conducted as 32 °C, pH: 6.2, stirring 250 rpm, under anaerobiosis. DMBI was supplemented after 72 h of fermentation. Results represent the mean of duplicates.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6596409/v1/e40ace324c86a5400d7df76c.png"},{"id":82809733,"identity":"9ea86ff1-b63f-4140-9fc9-dfa64e8b390e","added_by":"auto","created_at":"2025-05-15 13:13:24","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":899618,"visible":true,"origin":"","legend":"","description":"","filename":"OptmB12SM.docx","url":"https://assets-eu.researchsquare.com/files/rs-6596409/v1/8c8138d4c00b129497fc6089.docx"}],"financialInterests":"","formattedTitle":"Production of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 on a soybean agro-industrial waste-based medium: bioprocess optimization in STR bioreactors","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eVitamin B\u003csub\u003e12\u003c/sub\u003e is an essential micronutrient required for human metabolism and blood cell synthesis.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Its deficiency leads to serious disorders, such as sensory and motor disturbances, depression, dementia, and, eventually, anemia and death.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The recommended human vitamin B\u003csub\u003e12\u003c/sub\u003e intake to prevent deficiency ranges from 4 to 20 \u0026micro;g \u0026bull; day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for adults.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Peculiarly, humans rely almost entirely on animal-derived food (e.g. meat, fish, and dairy products) to obtain this micronutrient through conventional diets.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHowever, in the Anthropocene (the current geological age), industrial production of animal-based food has been proven to cause severe environmental impacts.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e The sector occupies 75 of Earth\u0026rsquo;s arable land and it is the major driver for deforestation and biodiversity loss while responding to a massive greenhouse gas emission (\u0026gt;\u0026thinsp;9.7\u0026nbsp;billion t CO\u003csub\u003e2\u003c/sub\u003e-eq \u0026bull; year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and 78 of all eutrophication of superficial waters worldwide.\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e According to the EAT Lancet Commission, a drastic shift toward more plant-based diets must happen in the near future to establish a truly sustainable food system and to avoid worsening scenarios of global warming and climate change.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this sense, plant-based products are already growing in popularity.\u003csup\u003e \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e \u003c/sup\u003e In Europe, the sales of plant-based foods (e.g. meat, milk, and other plant-based analogues) increased by 49 % beween 2018 and 2020 (\u0026euro; 2.4\u0026nbsp;billion vs. \u0026euro; 3.6\u0026nbsp;billion, respectively).\u003csup\u003e \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e \u003c/sup\u003e In the USA, the market share of the plant-based segment surpassed US\u003cspan\u003e$\u003c/span\u003e 10\u0026nbsp;billion in 2022 and it is expected to double by 2029.\u003csup\u003e \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e \u003c/sup\u003e In this context, fortification of plant-based products with vitamin B\u003csub\u003e12\u003c/sub\u003e is crucial to prevent nutritional deficiencies, since plants do not synthesize or accumulate this essential micronutrient.\u003csup\u003e \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e \u003c/sup\u003e \u003c/p\u003e \u003cp\u003eVitamin B\u003csub\u003e12\u003c/sub\u003e is an expensive micronutrient. According to the ChemAnalyst, vitamin B\u003csub\u003e12\u003c/sub\u003e is worth around 1500 U\u003cspan\u003e$\u003c/span\u003e per kg and its demand is expected to grow 5.45% in ten years\u003csup\u003e \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e \u003c/sup\u003e. Industrial production of vitamin B\u003csub\u003e12\u003c/sub\u003e is carried out exclusively through bioprocess due to its structural complexity.\u003csup\u003e \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e \u003c/sup\u003e The dairy \u003cem\u003ePropionibacterium\u003c/em\u003e (dairy PAB) are particularly interesting bacterium because it holds the GRAS status and has the natural ability to synthesize vitamin B\u003csub\u003e12\u003c/sub\u003e in considerable amounts.\u003csup\u003e \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e \u003c/sup\u003e Recently, \u003cem\u003ePropionibacterium freudenreichii\u003c/em\u003e subsp. \u003cem\u003eshermanii\u003c/em\u003e ATCC 13673 has been reported to produce vitamin B\u003csub\u003e12\u003c/sub\u003e in an inexpensive and available soybean agro-industrial waste.\u003csup\u003e \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e \u003c/sup\u003e \u003c/p\u003e \u003cp\u003eThe Liquid Acid Protein Residue of Soybean (LAPRS) is an industrial effluent obtained during the soybean protein isolation process, and it is discharged into the environment as waste, after treatment. It possesses a high biochemical oxygen demand (\u0026gt;\u0026thinsp;20,000 mg O\u003csub\u003e2\u003c/sub\u003e \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) due to its organic components (carbohydrates and soluble proteins), which turns it into a potential culture medium for bioprocess. In the study mentioned above, the LAPRS medium composition was optimized for vitamin B\u003csub\u003e12\u003c/sub\u003e biosynthesis by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 growing in agitating flasks. After the transfer of this bioprocess to bench STR bioreactors, appreciable amounts of vitamin B\u003csub\u003e12\u003c/sub\u003e were obtained (~\u0026thinsp;2 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), however, the optimal growth condition was yet to be found. \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBased on these considerations, the aim of this work was to optimize the vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 growing in bench stirred tank bioreactors using the LAPRS as medium culture. Different bioprocess variables were investigated including aeration strategy (anaerobiosis followed by microaerophilic conditions or pure anaerobiosis) and pre-saccharification of complex soybean oligosaccharides. Additionally, temperature and pH control were optimized through design of experiments (DoE) approach.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Microorganisms and LAPRS medium culture\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ePropionibacterium freudenreichii\u003c/em\u003e subsp. \u003cem\u003eshermanii\u003c/em\u003e ATCC 13673 strain was purchased from Andr\u0026eacute; Tosello Foundation (Campinas, SP, Brazil). The LAPRS was kindly donated by the International Flavors \u0026amp; Fragrances Inc. (Esteio, RS, Brazil) and collected and concentrated as described in \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The first LAPRS sample was used as substrate for preliminary experiments and the second and third LAPRS samples were used on the design of experiments (DoE) assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Chemical composition of LAPRS\u003c/h2\u003e \u003cp\u003eThe LAPRS medium composition was previously described and optimized \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, the standard LAPRS medium culture for vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 used in this work has the following composition: total sugars, 6\u0026ndash;8 % protein 1 % cobalt, 250 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Cysteine, 1,200 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 5,6-Dimethilbenzimidazole (DMBI), 30 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Total carbohydrates, proteins, and ashes were measured for all LAPRS samples. The methodologies applied were Fehling test, Kjeldahl, and dry ashing, according to the Adolfo Lutz Institute protocols.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e The pH was measured using a pH probe (PH0-14, KASVI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Inoculum and growth in stirred tank bioreactors (STB)\u003c/h2\u003e \u003cp\u003eThe inoculum and growth conditions were performed as previously described.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Briefly, cryopreserved \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 cell suspensions were cultured at 30\u0026deg;C for 72 h to obtain the inoculum (optical density\u0026thinsp;=\u0026thinsp;1 unit ABS 600 nm). The experiments were performed in 2 L stirred tank bioreactors (STB) Biostat\u0026reg; B Plus (Sartorius, Germany) operating with 1.5 L work volume, including 10 %(in volume) of inoculum. The LAPRS medium was sterilized in the cultivation vessel at 121\u0026deg;C, 15 min. Bioprocess standard controls were as follows: temperature, 30\u0026deg;C, pH 6.5 adjusted with NaOH 7 M and alternating aeration, in which the first 72 h of cultivation were carried out under anaerobiosis, followed by 96 h under microaerophilic conditions obtained by 0.5 vvm of air flow and 250 rpm using two Rushton turbines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Preliminary experiments\u003c/h2\u003e \u003cp\u003ePreliminary experiments were performed to establish some bioprocess variables prior the optimization trough DoE approach. Additional supplementation, pre-saccharification, and the selection of aeration strategy were investigated in these experiments.\u003c/p\u003e \u003cp\u003eCobalt and DMBI are key components of vitamin B\u003csub\u003e12\u003c/sub\u003e structure.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e In order to check whether their concentration would impact vitamin B\u003csub\u003e12\u003c/sub\u003e production in bioreactors, we carried out an experiment increasing their concentration by 20 %in relation to the standard medium described in the section 2.2.\u003c/p\u003e \u003cp\u003eThe carbohydrates present in the LAPRS are mostly complex soybean oligosaccharides.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Therefore, a pre-saccharification step was also investigated. For this reaction, based on Coghetto et al.,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e a combination of invertase (1,000 U \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and alpha-galactosidase (1,800 U \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were used at 50\u0026deg;C, pH 4.5 for 3 h, reaching a hydrolysis degree of ~\u0026thinsp;60 %. wo different aeration strategies are often applied for dairy PAB growth and vitamin B\u003csub\u003e12\u003c/sub\u003e production (i.e. anaerobiosis/microaerophilic conditions or pure anaerobiosis). Here, we compared them under the following conditions: 1) alternating anaerobiosis (72 h) followed by microaerophilic conditions (96 h); and 2) culture run exclusively under anaerobiosis (168 h).\u003c/p\u003e \u003cp\u003eThe experiment performed under standard conditions, as described in section 2.3., was coded as LA30. The experiments with additional supplementation, pre-saccharification, and exclusively anaerobiosis were coded as: LA30+, HA30, and HAN30, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Central composite design 2\u003csup\u003e2\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eAfter the initial tests, a central composite design 2\u003csup\u003e2\u003c/sup\u003e was applied to find the optimal temperature and pH values for vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 growing in the LAPRS based medium. The variables levels tested in this study are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The vitamin B\u003csub\u003e12\u003c/sub\u003e specific yield was chosen as response variable for statistical analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTemperature and pH levels studied in the central composite design 2\u003csup\u003e2\u003c/sup\u003e to optimize vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003eP. shermanii\u003c/em\u003e in STB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eLevel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1.41\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;1\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;1.41\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Biomass, vitamin B\u003csub\u003e12\u003c/sub\u003e, monosaccharides, and organic acids analyses\u003c/h2\u003e \u003cp\u003eBiomass was measured by the gravimetric method and the results were expressed as dry weight of cells. Organic acids were analyzed using HPLC-RID system equipped with Bio-Rad HPX-87H column and H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 5 mM solution as mobile phase. Monosaccharides (glucose, galactose, and fructose) were measured by HPLC system equipped with Bio-Rad HPX-87C column using Milli-Q water as mobile phase. Vitamin B\u003csub\u003e12\u003c/sub\u003e analysis was performed with a validated method previously described.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Briefly, 20 mL of broth were centrifuged, the cellular pellet was washed with distilled water and resuspended in 20 mL of extraction buffer (phosphate 0.1 M; KCN, 0.1 %weight fraction; pH 4.5). The extraction was performed in amber flasks, using autoclave (Phoenix, Brazil) at 121\u0026deg;C for 15 min. The extracts were centrifuged, and the supernatant was collected, filtered through acetate cellulose membranes (0.22 \u0026micro;m) and frozen (-18\u0026deg;C) until HPLC-DAD analysis using C18 column Shimadzu and a gradient elution of Milli-Q water:methanol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses (ANOVA and Tukey\u0026rsquo;s test) were performed using the GraphPad Prism version 9.5.1 (GraphPad software, USA). The mathematical modelling of the central composite design 2\u003csup\u003e2\u003c/sup\u003e was performed using the software Statistica (v. 10.0, StatSoft, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. LAPRS chemical composition\u003c/h2\u003e \u003cp\u003eThe LAPRS is a nutrient-rich agro-industrial residue. The chemical composition of the LAPRS samples used as culture medium in this work is shown in the Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Carbohydrates are the major component in the LAPRS (up to 8% m/v). Most of them are presented as sucrose, stachyose, raffinose, the main sugars of soybean by-products.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Therefore, the enzymatic pre-treatment using hydrolytic enzymes may be necessary to liberate the monosaccharides and improve the bioprocess and it was investigated in this study.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical composition of the LAPRS samples used as the culture medium for vitamin B\u003csub\u003e12\u003c/sub\u003e production.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAnalyses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eResults\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eLAPRS-industrial sample 1\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eLAPRS-industrial sample 2\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eLAPRS-industrial sample 3\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbohydrates (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProteins (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAshes (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eThe means with the same letters in the rows indicates no statistical difference (Tukey\u0026rsquo;s test, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eProteins are present in lower amounts than sugars (1\u0026ndash;2% m/v) in the LAPRS (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), but in adequate concentrations to enable microbial growth, without any nitrogen supplementation. This fact has been shown in a previous work, where the supplementation of nitrogen did not produce any effect on vitamin B\u003csub\u003e12\u003c/sub\u003e specific yields by \u003cem\u003eP. shermanii\u003c/em\u003e in batch fermentations using LAPRS-based medium culture.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e On the other hand, it is important to note that the specific supplementation of one aminoacid, cysteine, is necessary. Cysteine is involved in S-Adenosyl methionine (SAM) regeneration, which is an important co-factor for vitamin B\u003csub\u003e12\u003c/sub\u003e formation.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e The supplementation of LAPRS culture medium with cysteine proved to be positive for vitamin B\u003csub\u003e12\u003c/sub\u003e accumulation by the \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673, hence, it was kept in this work.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe LAPRS shows a mineral content of up to 1.6% m/v (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It includes minerals required for microbial development, such as potassium (K), phosphorus (P), manganese (Mn), and magnesium (Mg) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, cobalt (Co) is not present in the LAPRS, and its supplementation is required because it is a central component of the vitamin B\u003csub\u003e12\u003c/sub\u003e molecule structure. The acid pH (pH 4.5) is a characteristic of the LAPRS due to the protein precipitation at the isoelectric point.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Nonetheless, the pH was controlled automatically in all fermentations according to the experimental design.\u003c/p\u003e \u003cp\u003eIt is worth to mention that a significant variation (ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed within pH, carbohydrate and protein content within the LAPRS industrial samples (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This was expected since LAPRS is an agro-industrial by-product and its composition is vastly influenced by the vegetable matrix and the manufacturing practices. However, the observed variation did not affect the strain growth kinetics (e.g. biomass production or substrate consumption), which highlights the robustness of our bioprocess.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Preliminary experiments\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the growth kinetics of \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 in the standard LAPRS medium and in the same medium with 20% increase of vitamin B\u003csub\u003e12\u003c/sub\u003e precursors (i.e. cobalt and DMBI). The growth patterns of \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 were similar independently of the supplementation strategy, and there was no statistical difference between treatments on bioprocess yields (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The only exception it was the propionate titer that decreased from 9.61 g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 6.77 g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e between LA30 and LA30+, respectively. It had statistical significance (ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but did not impact the other yields.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults obtained on the optimization of vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003ePropionibacterium freudenreichii\u003c/em\u003e subsp. \u003cem\u003eshermanii\u003c/em\u003e ATCC 13673 using LAPRS based medium on STB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperiment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiomass\u003c/p\u003e \u003cp\u003e(g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate consumption \u003c/p\u003e \u003cp\u003e(g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePropionate\u003c/p\u003e \u003cp\u003e(g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAcetate\u003c/p\u003e \u003cp\u003e(g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVitamin B\u003csub\u003e12 \u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYields B\u003csub\u003e12 \u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(mg \u0026bull; g \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e biomass)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eQ\u003csub\u003eB12 \u003c/sub\u003e\u003c/p\u003e \u003cp\u003emg \u0026bull; (L \u0026bull; h) \u003csup\u003e\u0026minus;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLA30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLA30 +\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0. 09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHAN30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVAL32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.24 \u0026plusmn; 1.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eAll results correspond to the culture time of 168 h, except for propionate values that are related to samples taken at 72 h of growth. The means with the same letters in columns indicates no statistical difference (Tukey\u0026rsquo;s test, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). LA30: Experiment performed under standard conditions; LA30 +: Experiment performed with additional supplementation (20% increase of vitamin B\u003csub\u003e12\u003c/sub\u003e precursors); HA30: Experiment performed with hydrolyzed LAPRS and standard supplementation; HAN30: Experiment performed with hydrolyzed LAPRS, standard supplementation under anaerobiosis; VAL32: Experiment performed with hydrolyzed LAPRS, standard supplementation under anaerobiosis and optimized pH (6.5) and temperature (32\u0026deg;C) control.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePreviously, it was noticed that the transfer of this bioprocess using LAPRS-based medium from shaking flasks to bench STB increased the biomass yield of \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 up to 4 folds. On the other hand, the vitamin B\u003csub\u003e12\u003c/sub\u003e specific yield (Y\u003csub\u003eB12\u003c/sub\u003e) decreased from 0.6 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 0.25 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003csup\u003e20\u003c/sup\u003e In this context, we hypothesized that essential vitamin B\u003csub\u003e12\u003c/sub\u003e precursors could be lacking. However, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the additional supplementation did not increase the vitamin B\u003csub\u003e12\u003c/sub\u003e specific yield, which remained 0.24 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In this case, the selective increase in biomass yield can be attributed just to the better variables control in STB (the pH and alternation aeration) and not the lack of vitamin B\u003csub\u003e12\u003c/sub\u003e precursors. Therefore, the standard medium was kept in further experiments.\u003c/p\u003e \u003cp\u003eThe availability of easily fermentable sugars is another key factor in bioprocess. Since LAPRS based medium is rich in complex carbohydrates (stachyose and raffinose), the influence of a pre-saccharification process was also investigated. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows the growth kinetics of \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 on the hydrolyzed LAPRS medium. The hydrolysis at least doubled the substrate consumption (21.8 g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), vitamin B\u003csub\u003e12\u003c/sub\u003e titer (4.16 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and specific yield of B\u003csub\u003e12\u003c/sub\u003e (0.5 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), compared to LA30 experiment (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Unlike the other yields, propionate and acetate did not have significant changes between LA30 and HA30 experiments. This feature of the \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 is desirable for vitamin B\u003csub\u003e12\u003c/sub\u003e production since high amounts of propionate (\u0026gt;\u0026thinsp;10 g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) may slow down the cell growth or even stop it due to end-product inhibition.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMonosaccharides usually enables microbes a faster growth and metabolic activity due to less energy required for the substrate breakdown.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The higher metabolic flow produces more energy and the synthesis of vitamin B\u003csub\u003e12\u003c/sub\u003e precursors, thus increasing its yield.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Glucose and galactose were the sugars metabolized by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673, with negligible fructose consumption (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The preference for glucose rather than fructose by dairy PAB has been reported by Piwowarek et al.,\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e who show that \u003cem\u003eP. freudenreichii\u003c/em\u003e T82 was able to completely deplete glucose as substrate, but only half the amount of fructose under the same conditions and time. Based on our findings, the glucose and galactose were considered the total sugars in the afterwards experiments using the hydrolyzed LAPRS-based medium.\u003c/p\u003e \u003cp\u003eDifferent aeration strategies (alternating anaerobiosis/microaerophilic conditions and pure anaerobiosis) have been tested for vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003ePropionibacterium\u003c/em\u003e genus. To decide the best condition both strategies were evaluated for \u003cem\u003eP. shermanii\u003c/em\u003e ATCC13673 growth using the hydrolyzed LAPRS medium culture. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows the strain growth under alternating aeration (experiment HA30), while Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows the results for anaerobiosis (experiment HAN30).\u003c/p\u003e \u003cp\u003eThe aeration after 72 h had significant effect on biomass and acetate accumulation compared to the culture under anaerobiosis (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This phenomenon is typical of alternating aeration strategy. The oxygen triggers conversion of propionate to acetate and increases energy production by the strain, which leads to higher biomass and acetate production, as observed in the HA30 experiment.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e However, the alternating aeration strategy did not influence the volumetric production of vitamin B\u003csub\u003e12\u003c/sub\u003e compared to the exclusively anaerobic culture (4.16 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 4.00 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively). In fact, the anaerobiosis enabled \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 to reach a higher specific yield of vitamin B\u003csub\u003e12\u003c/sub\u003e compared to the alternating aeration strategy (0.71 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.50 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively).\u003c/p\u003e \u003cp\u003eMicrobial biosynthesis of vitamin B\u003csub\u003e12\u003c/sub\u003e follows two distinct routes, known as: aerobic and anaerobic routes.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Dairy PAB follows the anaerobic route and even low volumetric oxygen transference coefficient (K\u003csub\u003eL\u003c/sub\u003ea \u0026lt; 10 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at the early growth phase significantly affects the vitamin B\u003csub\u003e12\u003c/sub\u003e biosynthesis.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e On the other hand, these bacteria use oxygen at late growth phase for DMBI synthesis (essential to produce active vitamin B\u003csub\u003e12\u003c/sub\u003e) via Blub/CobT2 enzyme.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Additionally, aeration triggers propionate consumption and can attenuate end-product growth inhibition.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Hence, alternating aeration along the culture have been widely used in bioprocess for vitamin B\u003csub\u003e12\u003c/sub\u003e production with dairy PAB.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHowever, when DMBI is supplemented to the culture medium, it seems that the oxygen-dependent DMBI synthesis do not contribute significantly for vitamin B\u003csub\u003e12\u003c/sub\u003e production. Our findings suggest that under anaerobiosis, secured the external DMBI supplementation, dairy PAB can accumulate significantly more vitamin B\u003csub\u003e12\u003c/sub\u003e per gram of cells (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, anaerobiosis was chosen for vitamin B\u003csub\u003e12\u003c/sub\u003e production in the following experiments.\u003c/p\u003e \u003cp\u003eTemperature and pH are also important variables to investigate during the bioprocess development. They affect dairy PAB growth rate and vitamin B12 production. Different temperatures (28\u0026deg;C to 38\u0026deg;C) and pH (5 to 7) values have been considered for dairy PAB cultures.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Therefore, after the testing the previous bioprocess variables, the optimum values of these two parameters were investigated using the DoE approach.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Bioprocess optimization by Design of Experiments (DoE)\u003c/h2\u003e \u003cp\u003eA central composite design 2\u003csup\u003e2\u003c/sup\u003e was applied for the optimization of temperature and pH values. Ten independent experiments were performed according to the experimental design presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, including two replicates at the central point to estimate the error.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimental matrix of the central composite design 2\u003csup\u003e2\u003c/sup\u003e with two duplicates at central point and the vitamin B\u003csub\u003e12\u003c/sub\u003e specific yield produced by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 growing in STB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExperiment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eIndependent variables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eResponse variable\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eTemperature (\u0026deg;C)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eYields B\u003c/b\u003e\u003csub\u003e\u003cb\u003e12 \u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mg \u0026bull; g\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003edry cell)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe mathematical modeling of the results based on the experimental data was statistically significant (ANOVA, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and presented a satisfactory coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.89). The equation (Eq.\u0026nbsp;1) explained\u0026thinsp;~\u0026thinsp;90% of the vitamin B\u003csub\u003e12\u003c/sub\u003e biosynthesis by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 growing in STB.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEq.\u0026nbsp;1\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eYB\u003c/em\u003e \u003csub\u003e \u003cem\u003e12\u003c/em\u003e \u003c/sub\u003e \u003cem\u003e= -19.2161\u0026thinsp;+\u0026thinsp;1.7898A \u0026ndash; 0.1873A\u003c/em\u003e \u003csup\u003e \u003cem\u003e2\u003c/em\u003e \u003c/sup\u003e\u0026thinsp;\u003cem\u003e+\u0026thinsp;0.8959B \u0026ndash; 0.0156B\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;\u003cem\u003e+\u0026thinsp;0.0171AB\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe response surface obtained through the mathematical modeling is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Both variables presented a negative effect on vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Thus, maximum vitamin B\u003csub\u003e12\u003c/sub\u003e specific yields were observed around the lowest to the central levels tested, respectively pH 5.5 to 6.5, and temperature 30\u0026deg;C to 33.5\u0026deg;C. This finding is in accordance with previous reports for dairy PAB growth and propionate production as well.\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e The mathematical modelling predicted optimal vitamin B\u003csub\u003e12\u003c/sub\u003e specific yields (\u0026gt;\u0026thinsp;0.76 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) controlling pH and temperature at 6.2 and 32\u0026deg;C, respectively. The specific yields of vitamin B\u003csub\u003e12\u003c/sub\u003e obtained experimentally was 0.84 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, validating the mathematical prediction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Growth kinetics of \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 under optimized conditions\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the growth kinetics of \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 under optimized conditions (experiment VAL32). The peak of biomass and vitamin B\u003csub\u003e12\u003c/sub\u003e production were obtained at 72 h and 96 h of culture, respectively. It represents a significant increase in productivity compared to the first experiment LA30. In addition, the vitamin B\u003csub\u003e12\u003c/sub\u003e specific yields more than tripled compared to the same experiment (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, the biomass production and vitamin B\u003csub\u003e12\u003c/sub\u003e specific yield did not differ statistically from the previous experiment HAN30 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Yet, an increase trend was observed in both outcomes. Biomass went from 5.35 g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 6.72 g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, and vitamin B\u003csub\u003e12\u003c/sub\u003e specific yield from 0.71 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 0.84 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Additionally, the vitamin B\u003csub\u003e12\u003c/sub\u003e titer in the VAL32 experiment it was the highest of them all (5.64 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This indicates that the optimization of temperature and pH values improved \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 biomass production, at the same time increasing the vitamin B\u003csub\u003e12\u003c/sub\u003e synthesis.\u003c/p\u003e \u003cp\u003eVitamin B\u003csub\u003e12\u003c/sub\u003e production by dairy PAB fluctuates according to the strain, medium culture composition and other bioprocess variables, such as the culture technique applied (batch, perfusion bioreactor, extractive fermentation, among others).\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Under the conditions of the batch process, using synthetic medium, vitamin B\u003csub\u003e12\u003c/sub\u003e titer produced by these microorganisms usually ranges from 6 to 30 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with specific yields around 0.6 to 0.9 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of dry cells.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Using agricultural wastes as substrate, the outcomes of vitamin B\u003csub\u003e12\u003c/sub\u003e can be less expressive. Hedayti et al.,\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e obtained approximately 3 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of vitamin B\u003csub\u003e12\u003c/sub\u003e by \u003cem\u003ePropionibacterium freudenreichii\u003c/em\u003e PTCC1674 growing in rice bran substrate. Similar results were reported by Hajfarajollah et al.,\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e (2.74 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in a bioprocess using \u003cem\u003ePropionibacterium freudenreichii\u003c/em\u003e PTCC1674 and sunflower waste oil.\u003c/p\u003e \u003cp\u003eIn this work, after the optimization process, a considerable amount of vitamin B\u003csub\u003e12\u003c/sub\u003e was obtained (5.64 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) using the LAPRS medium culture. Moreover, the specific yields reached (0.84 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry cells) were as high as those reported for dairy PAB growing in synthetic media. This bioprocess based on agro-industrial waste and a GRAS microorganism can be an alternative to be used in the fortification of plant-based foods. The bioaccessibility of vitamin B\u003csub\u003e12\u003c/sub\u003e delivered by dairy PAB biomass has been proven to be as high as the pharmaceutical form of vitamin B\u003csub\u003e12\u003c/sub\u003e.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCommercial vitamin B\u003csub\u003e12\u003c/sub\u003e production uses refined substrates, non-food grade strains (i.e. \u003cem\u003ePseudomonas denitrificans)\u003c/em\u003e and requires expensive downstream processing to obtain the highly purified pharmaceutical vitamin B\u003csub\u003e12\u003c/sub\u003e\u003csup\u003e31\u003c/sup\u003e. On the other hand, in this work was utilized a cheap and highly available soybean agro-industrial waste as substrate. Additionally, \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 is a GRAS microorganism whose biomass could be added directly to plant-based food products to fortify them, without the expensive downstream steps (e.g. cell lysis, crystallization \u003cem\u003eetc\u003c/em\u003e.). This unique combination, coupled to the high vitamin B\u003csub\u003e12\u003c/sub\u003e yields obtained after optimization, may reduce costs for vitamin B\u003csub\u003e12\u003c/sub\u003e fortification of plant-based foods and highlights the importance of the bioprocess developed in this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe vitamin B\u003csub\u003e12\u003c/sub\u003e production of \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673 grown in STB using LAPRS as medium culture was successfully optimized, being a new, cheap, and safe alternative to produce this important vitamin using exclusively plant-based medium. The enzymatic hydrolysis of complex carbohydrates present in LAPRS enabled the strain to produce considerable amounts of vitamin B\u003csub\u003e12\u003c/sub\u003e. Concerning aeration, anaerobiosis was the most suitable strategy for vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673. Our results suggest that, when DMBI supplementation is secured, an alternating aeration strategy may not be required for active vitamin B\u003csub\u003e12\u003c/sub\u003e synthesis by dairy PAB. Finally, DoE approach allowed us to find the optimal temperature, and pH control was established for efficient biomass production (6.72 g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and vitamin B\u003csub\u003e12\u003c/sub\u003e yields (5.64 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.84 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry cell) by \u003cem\u003eP. shermanii\u003c/em\u003e ATCC 13673, whose biomass could be used to fortify plant-based products in the food industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eWork’s Funding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq, Brazil), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, RS), through grants from Project RITES TO 22/2551-0000397-4, and scholarships from CAPES Code Fund 001 (Brazil).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD. A. A.: \u0026nbsp; Conceptualization; design of experiments; bench work; writing the original manuscript.\u003c/p\u003e\n\u003cp\u003eM. A. Z. A. Supervision (D. A. A.), Conceptualization; design of experiments; funding providing, writing and revising the original manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors of this work declare to have no conflicts of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be available upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNielsen, M. J., Rasmussen, M. R., Andersen, C. B. F., Nex\u0026oslash;, E., \u0026amp; Moestrup, S. K. (2012). Vitamin B12 transport from food to the body\u0026rsquo;s cells - A sophisticated, multistep pathway. \u003cem\u003eNature Reviews. 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Bioaccessibility of vitamin B12 synthesized by Propionibacterium freudenreichii and from products made with fermented wheat bran extract. \u003cem\u003eCurr Res Food Sci\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e, 499\u0026ndash;502. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.crfs.2021.07.009\u003c/span\u003e\u003cspan address=\"10.1016/j.crfs.2021.07.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sustainability, agro-industrial residues, plant-based foods, cobalamin, bioprocess technology","lastPublishedDoi":"10.21203/rs.3.rs-6596409/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6596409/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe production and use of plant-based foods are increasing based on health, ethical, and environmental concerns. However, plants do not synthesize vitamin B\u003csub\u003e12\u003c/sub\u003e, thus the fortification of plant-based foods is highly recommended to prevent its deficiency. The aim of this work was to optimize vitamin B\u003csub\u003e12\u003c/sub\u003e production by \u003cem\u003ePropionibacterium freudenreichii\u003c/em\u003e subsp. \u003cem\u003eshermanii\u003c/em\u003e ATCC 13673 growing on stirred tank bioreactors using the liquid acid protein residue of soybean, an agro-industrial waste, as medium culture. The influence of medium supplementation, pre-saccharification of sugars, and aeration strategies were investigated. The pH and temperature control were optimized applying the Design of Experiments (DoE) approach, using a central composite design. After the optimization process, the vitamin B\u003csub\u003e12\u003c/sub\u003e production more than tripled (from ~\u0026thinsp;1.5 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e up to 5 mg \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Further, cultures produced high biomass concentrations for this bacterium (\u0026gt;\u0026thinsp;6 g \u0026bull; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), whilst increasing by three-fold the specific biomass yield of vitamin B\u003csub\u003e12\u003c/sub\u003e (\u0026gt;\u0026thinsp;0.8 mg \u0026bull; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry cell). This bioprocess based on cheap and available soybean agro-industrial waste and a GRAS microorganism can be an efficient alternative source of vitamin B\u003csub\u003e12\u003c/sub\u003e production to fortify plant-based products in the future.\u003c/p\u003e","manuscriptTitle":"Production of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 on a soybean agro-industrial waste-based medium: bioprocess optimization in STR bioreactors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 13:13:19","doi":"10.21203/rs.3.rs-6596409/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-05-13T12:22:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T08:51:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2025-05-08T07:37:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2025-05-05T13:19:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"65bc3c71-8560-4a9d-89e7-850ecd861c32","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:06:32+00:00","versionOfRecord":{"articleIdentity":"rs-6596409","link":"https://doi.org/10.1007/s12010-025-05402-1","journal":{"identity":"applied-biochemistry-and-biotechnology","isVorOnly":false,"title":"Applied Biochemistry and Biotechnology"},"publishedOn":"2025-11-15 15:57:22","publishedOnDateReadable":"November 15th, 2025"},"versionCreatedAt":"2025-05-15 13:13:19","video":"","vorDoi":"10.1007/s12010-025-05402-1","vorDoiUrl":"https://doi.org/10.1007/s12010-025-05402-1","workflowStages":[]},"version":"v1","identity":"rs-6596409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6596409","identity":"rs-6596409","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}