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Additional file 1: Figure S1. Colony morphology used to assess strain-specific colony forming units in co-cultures. P. freudenreichii NCC 1177 on LPD agar (A); B. amyloliquefaciens NCC 156 on TSB agar; L. paracasei subsp. paracasei NCC 2511 on LPD agar (C). Figure S2. Time resolved changes of dissolved oxygen and pH value during aerobic growth on sunflower seed milk. The data comprise cultures using P. freudenreichii NCC 1177 (A), L. paracasei subsp. paracasei NCC 2511 (B), B. amyloliquefaciens NCC 156 (C), and a co-culture of two strains (D). n=1. Figure S3. Co-cultivation of P. freudenreichii NCC 1177 and B. amyloliquefaciens NCC 156 in UHT-processed sunflower seed milk. The data comprise colony forming units (A), the content of vitamin B12 (B), and vitamins B3, B6, and B7 (C), the relative amount of favored and unfavored volatile, inferred from the total peak area of GC/MS-based volatile analysis (D), the level sucrose, raffinose, and stachyose (E), the level of extracellular l-lysine, l-leucine, l-tryptophan, and l-methionine (F), the level of acetoin, 2,3-butanediol, propionate, and acetate (G), and the protein score PDCAAS (H). n=3. Table S1. Strain specific pre-culture conditions. As media, Mann-Rogosa-Sharpe medium (MRS) and modified tryptic soy broth (TSB) were used. Regarding oxygen supply, strains of P. freudenreichii were grown under anaerobic conditions. L. paracasei subsp. paracasei NCC 2511 was grown under microaerobic conditions, and B. amyloliquefaciens NCC 156 was grown aerobically. All strains were grown at 30 °C. Table S2A. Growth and vitamin B12 production of P. freudenreichii NCC 1177 on sunflower seed milk: Impact of different supplements added to the process. The incubation in the supplemented plant milk was carried out at 30 °C for 72 hours, including an initial anaerobic phase (48 hours), followed by an aerobic phase (24 hours). In addition, a non-supplemented process was conducted as control. The plant milk was pasteurized prior to cultivation. The vitamin B12 level and the cfu number reflect the final values at the end of the fermentation. n=3. Table S3. Metabolic profile of P. freudenreichii NCC 1177, B. amyloliquefaciens NCC 156, and L. paracasei subsp. paracasei NCC 2511 after aerobic and anaerobic growth on pasteurized sunflower seed milk. The fermentation was carried out at 30 °C either anaerobically (48 hours) or aerobically (24 hours). In addition, the composition of the milk at the start (including the inoculum) is given. For each parameter, the maximum absolute concentration change (increase or decrease), observed among all conditions, is highlighted in yellow. For the representation of the data as relative changes, this maximum change was normalized to a value of 1. The change of the other conditions was normalized to this maximum (Fig. 3). The data represent the final values under each condition. n=3. Table S4. Growth and vitamin B12 production during co-culturing of P. freudenreichii NCC 1177 and B. amyloliquefaciens NCC 156 in pasteurized sunflower seed milk: Impact of inoculum level and process conditions. In different set-ups, strain NCC 1177 was inoculated at a 10-fold, 100-fold, and 1,000-fold higher level than strain NCC 156. In all cases, the total inoculum of both strains was 2 × 107 cfu mL-1. Regarding process operation, one set-up comprised first a 24 h aerobic phase, followed by a 48-h anaerobic phase, whereas the two phases were reverted in a second set-up. All fermentations were carried out at 30 °C. The plant milk was pasteurized prior to fermentation. Vitamin level and cell growth display the final values after 72 h. n=3. Table S5. Dynamics of free amino acids during co-culturing of P. freudenreichii NCC 1177 and B. amyloliquefaciens NCC 156 in UHT-treated sunflower seed milk. The process involved a 48-h anaerobic phase, followed by a 48-h aerobic phase. n=3. |
