## 4 Discussion
intermedia_ resulted in a reduction in the fat content of the product, from 9.1 to 7.7 g/100 g DM [35]. A similar trend was observed in tempe fermentations with _Rhizopus oligosporus_[36], where the growth of the strain in the lower-fat substrate (white quinoa) resulted in an enrichment of fat in the product, while the opposite relationship was noted for the more fat-rich coloured quinoa. [PERSON] et al. [32] reported a decrease in starch content after fermentation of leftover bread with _N. intermedia_. In the case of bread fermentation in our experiments, an increase in the level of reducing sugars was observed, from seven- to eleven-fold. Amyloidic enzyme activity was detected by [PERSON] after 48 h of _N. intermedia_ fermentation on a typical oncom substrate of peanut press cake [37], as well as by [PERSON] et al. [38] in the conditions of submerged fermentation. From the results obtained in both experiments, it is clear that the amount of vitamin B12 determined in oncom products depended significantly on the strain of _P. freudenreichii_, on the CFU dose used as inoculum, but also significantly on the type of culture medium--in this case, stale bread. The number of bacteria determined after 5 days of culture in both experiments was lower than the amount introduced into the fermentation substrate. This either means that _P. freudenreichii_ had unfavourable conditions for growth or that after such a long time (5 days) the culture went into the die-off phase. A significantly higher amount of CFU was determined in the second experiment regardless of the type of substrate used. It is  
Figure 1: Examples of mixed bread oncom products from Experiment I: control on the left, c-f 1 on the right (photo: [PERSON]). known, however, that these bacteria poorly proliferate when growing in a solid medium. Additionally, their growth depends on ensuring anaerobic conditions and optimal pH, which typically oscillates around 7.0 [19]. The higher levels of vitamin B12 determined when Okin bread was used as a fermentation substrate compared to mixed bread, may have been related to a more favourable pH value at the start of the culture (5.7 vs. 4.3), but also a higher content of sugars (1.76/100 g DM vs. 1.08/100 g DM). Bacteria from the species _P. freudenreichii_ primarily utilize carbohydrates as a carbon source, including glucose, lactose, fructose, ribose, and galactose, as well as organic acids such as lactic acid [19]. [PERSON] et al. [24] pointed out the significant influence of substrate pH during fungal-bacterial co-fermentation aimed at enriching tempe with vitamin B12. Attempts at simultaneous co-fermentation of _Rhizopus oligosporus_ and _P. shermanii_ with the addition of _Lactobacillus casei_ resulted in less success, leading to the maintenance of acidic pH over the period of time. In the cited work, vitamin B12 was determined in tempe at a level of 0.18 ug/100 g DM [24]. Significantly higher levels of the vitamin in tempe from lupine were obtained by [PERSON] et al. [26], at 1.2 ug of the vitamin in 1 g DM. The authors ensured that the initial pH of the fermentation substrate exceeded 5 (5.3-5.5), and after cultivation, the pH increased to a level of approximately 7.0. This increase in pH may have promoted the proliferation of _P. freudenreichii_ and the production of vitamin B12. Comparing, in turn, the density of the bacterial inoculum, it must be said that [PERSON] et al. [26] introduced significantly more CFU into the substrate than in our experiments. Values of vitamin B12 in the produced tempe from lupin at levels similar to oncom from Okin bread (dose of 10\({}^{9}\) CFU/g substrate) were obtained by [PERSON] et al. [25] using the same _P. freudenreichii_ strain DSM 20271 (0.97 vs. 1.3 g/100 g fresh weight). Despite initially using a CFU dose two orders of magnitude smaller than in our experiment, these authors likely observed bacterial proliferation due to the more favorable pH for bacterial growth (6-7) or a shorter fermentation time.