3.2.4. Microstructure

Figure 3 reports the SEM micrographs of cross sections of raw pasta (PC, P5, P10, and P15). Starch granules were well visible in all the samples, and the protein matrix did not appear disrupted. However, in P10 and P15, starch granules seem to be immerged in a more dense and compact structure. Moreover, P10 and P15 exhibit the presence of a reticulated structure, probably formed from the interaction of inulin with the protein network, which could have strengthened the pasta structure and thus giving a higher firmness to the cooked pasta as observed by the instrumental analysis.

**Figure 2.** Sensory characteristics of control (PC) and inulin-enriched (P5, P10, P15) fresh pasta. Values are expressed as mean ± standard deviation; different letters for each parameters mean significant statistical differences (*p* < 0.05) to one-way ANOVA followed by Tukey's HSD test.

**Figure 3.** SE-SEM micrographs of control (PC) and inulin enriched (P5, P10, P15) fresh pasta (white bar = 20 μm).


Table 3 reported the proximate composition of fresh pasta. Significant differences were found among the samples for all the parameters considered. Specifically, inulin-enriched pasta showed a decline in protein content compared to the control (PC) due to a rise in total dietary fiber, which reached values of 3.44 g/100 g in P5, 8.16 g/100 g in P10, and 12.41 g/100 g in P15, exceeding the data observed by Padalino et al. [71] in pasta fortified with tomato byproducts. Therefore, the results in terms of total dietary fiber allow for labelling P5 as a "source of fibre", while P10 and P15 could be labelled as a pasta having "high fibre content" [72], according to Reg. (EU) 1924/2006, enhancing the nutritional value of fresh pasta. Moreover, higher ash and lower lipid content were found in P10 and P15 than P5 and PC.


**Table 3.** Proximate composition of fresh pasta (g/100 g).

PC, control pasta without inulin addition; P5, pasta with 5% of inulin added; P10, pasta with 10% of inulin added;P15, pasta with 15% of inulin added. The values represent means of triplicates ± standard deviation; different letters indicate significant differences; different letters in the same column mean significant statistical differences (*p* < 0.05) to one way ANOVA followed by Tukey's HSD test.

### 3.3.2. In Vitro Starch Hydrolysis

In consideration the pivotal role of nutritional aspects for modern consumers and in recent scientific research aimed at producing foods with a lower glycemic index [73,74], cooked fresh pasta samples were analyzed for in vitro starch hydrolysis. As shown in Figure 4, the addition of AR inulin in fresh pasta promoted the decrease of HI and pGI. On average, the HI and pGI values of fresh pasta samples enriched with AR inulin (P5, P10, and P15) were statistically lower than the control (PC). Specifically, P5 reached a significantly (*p* < 0.05) lower value of pGI compared to the control (PC): 63.01 and 66.54, respectively. However, a higher concentration of AR inulin resulted in a further decrease of HI and pGI, as shown in P10 and P15. In fact, P10 has a statistically lower pGI value (58.42) compared to both the control (PC) and P5. Nevertheless, no significant differences were observed for P10 and P15 samples in terms of pGI and HI. Data demonstrate a lower pGI response following ingestion of AR inulin-enriched fresh pasta compared to the control.

**Figure 4.** Results of hydrolysis index (HI) and predicted glycemic index (pGI) of control (PC) and inulin-enriched (P5, P10, P15) fresh pasta. The values represent means of triplicates ± standard deviation; different letters indicate significant differences (*p* < 0.05) according to one-way ANOVA followed by Tukey's HSD test.

Inulin, which is not digestible by humans, has interesting properties as a source of fermentable energy for some intestinal bacteria that produce short-chain fatty acids, which are essential for maintaining the intestinal homeostasis. In addition to stimulating digestion

and regularity of intestinal transit, inulin may favor the presence of *Bifidobacterium* in the microbiota and, at the same time, decrease harmful bacteria [75,76]. Several studies have highlighted the beneficial role of inulin also in glycemic response [41,77–79]. Inulin—as a soluble fiber—helps keep blood sugar under control, since the fibers present in complex carbohydrates take longer to release the sugars present in the body: the slow release of glucose prevents glycemic peaks both upwards and downwards by balancing the energy intake and limiting the accumulation of fat due to the excess insulin [31,80]. The gelling effect of these fibers causes the formation of a film on the walls of the stomach and intestines with consequent lower absorption of fats and sugars [81]. In contrast to a previous study on chicory inulin-enriched pasta (from 2.5% to 10%) that did not reduce the pGI compared to the control [28], our results confirmed that AR was able to reduce the pGI and HI already at 5% of its concentration, due to the high DP of inulin used in our pasta samples.

3.3.3. Evaluation of Effects Exerted by AR Inulin, Prebiotics, and Pathogen on Probiotic Growth

Herein, the prebiotic activity of inulin-enriched pasta was assessed in vitro in terms of probiotics growth. Furthermore, the inhibition of *E. coli* when co-cultured with probiotics was also determined. Compared to batches not containing carbohydrates (FM), the addition of pasta not containing inulin (FMPC) was sufficient to increase (~0.5 log10 CFU/mL) the cell density of all tested probiotics (Figure 5). This reflects the prebiotic contribution of fructans and arabinoxylans, which are non-digestible oligosaccharides naturally occurring in wheats [82], since their absence in gluten-free diets seems to affect the host microbiome and metabolome [83,84]. However, the presence of 3 g/L of inulin in FMP15 was able to further increase (>0.5 cycle) the cell density by 50% compared to the used probiotics (11 out of 22), while six strains (~27%) showed an increased cell density higher than one cycle. Besides the growth of probiotics, the acidification degree in batches followed the inulin concentration in a dependent manner. Values of ΔpH were, on average, 0.07 ± 0.06, 0.14 ± 0.11, and 0.20 ± 0.17 for FMP5, FMP10, and FMP15, respectively. No significant differences were found comparing the cell densities of *E. coli* in FMP to those of FMP5 (Figure 5). Oppositely, more than 50% (12 out of 22) of used probiotics were able to significantly decrease the cell density of *E. coli* in batches containing 3 g/L of inulin (FMP15). Meanwhile, ~36% of probiotics (eight strains) significantly decreased the *E. coli* cell density in batches containing 2 g/L of inulin (FMP10).

These results are in line with those previously stated by Kareem and co-workers [85], who reported that the combination of probiotics with prebiotics in vitro exhibited a grea<sup>t</sup> inhibition of pathogens due to a synergistic effect. Mechanisms based on microbial crossfeeding are largely distributed within the intestinal lumen [86]. A previous study concerning β-glucans-enriched pasta determined that 3 g of daily fiber supplementation was optimal to increase the saccharolytic metabolism in terms of SCFA profiling and improving the endothelial reactivity in healthy volunteers [87]. Therefore, although some used strains did not directly decrease *E. coli* growth, evidence suggests that additional taxa belonging to the human gu<sup>t</sup> microbiota (e.g., clostridial cluster IV, XIVa, and *Bifidobacterium*) can support the metabolism of inulin in SCFA in vivo, eliciting a boosted effect that contributes to the host's intestinal homeostasis [88].

**Figure 5.** In vitro prebiotic assay showing the cell density of 22 probiotic lactic acid bacteria cocultured with *E. coli* in fecal batches not containing carbohydrates (FM) or made with the addition of pasta without inulin (FMPC) and inulin-enriched pasta at 5 (FMP5), 10 (FMP10), and 15% (FMP15). "£" means >0.5 log10 cycle increased cell density (CFU/mL) of probiotic lactic acid bacte-ria in FMP15 versus FMP; "££" means >1 log10 cycle increased cell density (CFU/mL) of probiotic lactic acid bacteria in FMP15 versus FMP. "#" and "\$" indicate those probiotics that had respective-ly determined a significant decrease of *E. coli* cell density in FMP15 or both FMP15 and FMP10. "\*": significant decrease of *E. coli* cell density compared to FMP; "ns": no significant differences.
