3.4.1. Method Validation

The results of the prebiotic potential method validation trial are shown in Figure 2. The assay was successful in demonstrating noticeable changes in growth both with and without the presence of additives in its media. While the higher concentrations of the reference probiotic led to higher optical density readings after 72 h of incubation, it is interesting to note that this trend was not constant throughout the intermediate measures. Growth readings after 24 h suggested a preference for lower or null concentrations of inulin, which is likely associated with a breach in its maximum tolerable presence for this organism. Prolonged incubation revealed a reverse trend, with the growth of organisms under higher concentrations overtaking those previously mentioned. It is possible that inulin concentrations somewhere above 0.05%, together with the tested concentration of MRS media, provided an overabundance of soluble sugars, resulting in unfavourable osmotic pressures. These stressors were eventually overcome by the organism, which then made use of the higher abundance of nutrients to surpass the growth in the other conditions. Since these dynamics were easily verified in the conditions tested here, the testing of probiotic growth effects was carried on using this method.

**Figure 2.** Growth of *Lactobacilus delbrueckii* under the effect of rising concentrations of inulin. Each point represents the average of at least three determinations ± standard error.

### 3.4.2. Growth Effect on Probiotic Cultures

Figure 3 shows the influence of the digested spent *Aurantiochytrium* solution (sample) and the digestion blank (vehicle) in the growth of *B. bifidum*, *W. cibaria*, and *L. delbrueckii* via a measure of optical density. For the first two microorganisms mentioned, the presence of the digested sample was favourable in promoting their growth, although the kinetics of each revealed differences that may influence the effectiveness of this sample as a prebiotic agent. The presence of digested defatted *Aurantiochytrium* solution had an immediate benefit on the growth of *W. cibaria*, as can be verified in the OD recorded after 24 h. This sudden growth appeared to consume nearly all the available substrate, and no further changes, worth noting, in cell density were observed over the course of the assay. *Weissela cibaria* has proven to be fastidious when compared to other lactic acid bacteria (LAB) [51]. Its accelerated growth kinetics and adaptability to harsh mediums has led to an increased interest in using it as a majority culture in sourdough starters [51]. *Bifidobacterium bifidum* revealed an equally favourable outcome for the probiotic growth enhancement potential of

the digested defatted *Aurantiochytrium* solution, having reached 72 h of incubation with a significant DO difference between this sample and its digestion blank and growth control. In accordance with the features of a less fastidious anaerobic culture, the steepest increase in growth was observed between 24 and 48 h past medium inoculation, contrasting with the immediate spike in growth seen with *W. cibaria* immediately at 24 h. In contrast to the results commented so far, the growth of *L. delbrueckii* was unaffected by the presence of the digested defatted *Aurantiochytrium* sample or the digestion solution (vehicle). The scarcity of information regarding the non-lipid fraction of *Aurantiochytrium* sp. makes it difficult to point to specific causes for the selectivity of its prebiotic potential. Several studies testing the prebiotic potential of select compounds have also reported relatively lower growth of *L. delbrueckii*, but the authors did not establish likely causes [16,52,53]. Further studies of the non-lipidic fraction of *Aurantiochytrium* sp. might reveal hints about the selectivity of its prebiotic activity.

**Figure 3.** Growth of the probiotic strains *Bifidobacterium bifidum*, *Weissella cibaria,* and *Lactobacillus delbrueckii* under the presence of the digested defatted *Aurantiochytrium* sp. (Sample), the enzymatic digestion mixture (Vehicle), and the negative control saline solution (0% inulin). Each point represents the average of at least three determinations ± standard error. \* and \*\* group measures are statistically identical within the same time point (either 24, 48, or 72 h).

The differences in kinetics between *W. cibaria* and *B. bifidum* are unlikely to reflect in a significant manner within in vivo conditions, given the extensive amount of biotransformations induced by digestion, as well as the influence of the remaining gu<sup>t</sup> microbiota [19]. Selective or delayed growth of different gu<sup>t</sup> bacteria is a hot topic of current-day prebiotic research, but the results shown here are more indicative of the differences between the growth characteristics of the studied strains rather than conclusive evidence of the selectivity of the substrate [54]. The results do show a significant increase in the growth of both *W. cibaria* and *B. bifidum* under the presence of digested spent *Aurantiochytrium* sp. Given the comparatively high amount of fibre content revealed by the chemical analysis, it is safe to say that this biomass, currently seen as industrial waste, may hold potential as a prebiotic supplement, with a stronger endorsement depending on further studies.
