**4. Discussion**

In this study, a new extract from brewer's spent grains was used as a bioactive ingredient for food supplements and tested for its effects on postprandial glycemia and insulinemic response in a monocentric, randomized, cross-over, double-blind, placebocontrolled clinical trial.

Although the literature data report that the main fiber families present in BSG are insoluble fibers (i.e., cellulose, lignin, and hemicellulose) [19], the BSG extract used in this study does not contain insoluble fiber. This composition is probably due to the patented enzymatic extraction method that allows enrichment of the extract with soluble fibers [35]. In agreement with the literature data [19], this BSG extract consists of resistant starch (14.6 g/100 g), followed by AXs (7.5 g/100 g) and β-glucans (1.9 g/100 g). Among AXs, 5% and 95% of the total AX content are represented by high molecular weight and low molecular weight AXs, respectively. Moreover, the water extractable AXs represent about 16% and alkali extractable AXs represent about 84% of total AX content. The major gelling properties, which, in turn, are responsible for the lower and slower absorption of glucose from the diet, are mainly ascribed to water-soluble, high molecular weight AXs. Based on the obtained results regarding AXs, we expect the BSG extract effect on glucose absorption to be modest.

On the basis of the results of the analyses of the different kind of fiber of BSG extract, which was found to contain about 31 g of soluble fiber per 100 g of BSG extract, one stick pack of the BSG extract-based food supplement, at 5.0 g/stick pack of BSG extract, contains about 1.5 g of soluble fibers and 0.75 g of inulin (about 7% of the recommend consumption of 25 g for adult women and 38 g for adult men, based on epidemiologic studies showing protection against cardiovascular disease) [36].

Most of the phenolic compounds present in barley grains are found in the husk, and thus BSG represents a rich source of polyphenols. The TPC obtained from the analysis of BSG extract before the in vitro simulated digestion process resulted to be 0.49 g GAE/100 g. This value is comparable to those reported by Birsan et al. that found that TPC of the crude extract ranged from 0.28 to 0.38 g GAE/100 g, and by Meneses et al. that reported that TPC of the BSG extracted with water resulted to be 0.39 g GAE/100 g [37,38]. After the in vitro simulated digestion process, TPC was found to be 1.16 g GAE/100 g. Although this result is an overestimation of polyphenol content, it is however indicative of the presence of bioaccessible polyphenols that are not degraded by digestion and that can perform their beneficial effects at the intestinal level.

Ferulic acid is the main free and bound polyphenol in barley, malt, and BSG [39]. As ferulic acid has been shown to exert anti-diabetic effects in many in vitro and in vivo studies through different mechanisms of action (i.e., reduction of oxidative stress in pancreatic islets, which, in turn, causes their necrosis leading to reduced secretion of insulin, improvement in the activities of antioxidant enzymes (i.e., superoxide dismutase and catalase) in the pancreatic tissue, and increase of glucose uptake in insulin resistant cells) [40], to understand how much ferulic acid remains available to exert its beneficial biological activities after digestion, the residual free ferulic acid content after in vitro simulated oralgastric-duodenal digestion was determined. Ferulic acid concentration was found to be about 90 mg/100 g BSG extract. The ferulic acid content of BSG depends on the varieties of barley used, the malting and brewing processes, and the extraction method used to treat the sample before analysis. Mussatto et al. showed that by applying optimized alkaline hydrolysis conditions for the extraction of ferulic acid from BSG, its concentration resulted to be 9.65 mg/ per gram of solubilized lignin, corresponding to 286 mg/100 g of BSG [41]. The result obtained from the analysis performed in the present investigation showed a lower concentration of released ferulic acid in comparison with the result obtained by Mussatto et al. This result can be due to variations in the treatment of the samples that lead to different yields in the release of ferulic acid. Nevertheless, we decided to apply a simulated in vitro digestion process as it is more in line with the human digestive process and can give a more accurate view of the bioaccessibility and bioavailability of ferulic acid.

Regarding the results of the clinical trial on postprandial glycemia and insulinemia, a large body of evidence suggests that postprandial hyperglycemia is a risk factor for the onset of diabetes and cardiovascular disease [42,43].

The results of this clinical trial showed that blood glucose values measured after BSG extract-based food supplement intake were significantly lower than the corresponding values for the placebo group only in the descent phase of the glucose peak, respectively, after 90 min and 120 min, while at the baseline (t0) and in the first 60 min (t1–t3), the two glycemic curves overlapped substantially. We justify the absence of an effect of the intake of the food supplement taken with the standard meal on post-prandial blood glucose recorded in the first 60 min, with the low content and poor gelling properties of soluble fibers, especially AXs, present in BSG extract, that do not induce a significant reduction in glucose absorption. In addition, the glycemic curves of the subjects who took a placebo have trends typical of those curves recorded after the consumption of a starchy food. In fact, as reported by Brand-Miller et al. [44], starchy foods provide more glucose than sugary foods (i.e., soft drinks and fruit juices), and therefore were generally more likely to produce a curve that remained above baseline at 120 min. The intake of BSG extractbased food supplement, while not inhibiting the absorption of glucose, in the first 60 min, probably slightly reduces and slows down the absorption of glucose in accordance with the fiber content of the food supplement. The explanations of the modest recorded effect on postprandial glycemia produced by BSG extract based-food supplement may be due at least in part to the presence in the BSG extract of simple sugars, which increase glycemia, and to the fact that the effect of the food supplement is compared with the placebo that contains 15% inulin, which is a dietary fiber known to have hypoglycemic effects [45,46].

However, since the purpose of the clinical study was to test the effect of BSG extract, to exclude the effect of inulin used as carrier agent, the same amount of inulin present in the food supplement was added to the placebo. Although modest, the intake of the BSG extract based-food supplement produced a beneficial effect on glycemia as postprandial glycemic curve returned to baseline earlier. This improved clinical outcome was corroborated by significant reductions in postprandial insulinemic response. In particular, the blood insulin values of the subjects that took the food supplement were significantly lower from the first 15 min, with growing differences that reached maximum difference at 120 min from the first blood sample (t0). The mean insulin iAUC of the subjects who took the BSG extractbased food supplement was 19.7% significantly lower than the iAUC of the subjects taking placebo (*p* < 0.05). On the whole, these results mean that acute intake of BSG extract-based food supplement induces an improvement in postprandial insulinemic response. Moreover, it is worth noting that although the mean values of glycemia, BMI, and blood lipids of the recruited subjects are normal, however, on the basis of fasting glycemia, insulinemia, and triglyceridemia, most subjects showed a mild insulin resistance, as evidenced by HOMA Index and TyG values higher than 2.5 and 4.5, respectively. Thus, BSG extract-based food supplement, while not leading to a lowering of the postprandial glycemic curve, improved insulinemic response in subjects with mild insulin resistance.

Moreover, in accordance with the literature data, we found blood glucose values were significantly higher in men than in women irrespective of both the six measures the two experimental treatments, although these differences have no clinical relevance [47–50], and blood glucose tended to increase with age, independently from gender. It is well known that aging is associated with increased fasting blood glycemia due to a reduction in glucose-induced insulin release, and increased inflammation markers, which, in turn, increase insulin resistance in muscle and adipose tissue [51].

This work has limitations and strengths. The main limitations are represented by the fact that, due to the acute nature of this clinical trial, the effect of continuous ingestion of BSG extract and the influence of its fermentation products on glucose metabolism could not be determined, making it impossible to learn about any longer-term effects of this supplementation. Secondly, its effect on diabetic and pre-diabetic patients is unknown, as the subjects of this clinical trial were limited to normo-glycemic subjects showing just

mild insulin resistance. Finally, the presence in the food supplement of simple sugars, which together with the standard meal contribute to the rise of postprandial blood glucose, and the absence of an equal number of simple sugars in the placebo probably cause an underestimation of the actual properties of BSG extract-based food supplement to reduce the increase in post-prandial glycemia.

On the other hand, the major strength of this study is that the fiber and polyphenol composition of the BSG extract-based food supplement is known, and therefore the recorded effects on postprandial blood glucose and insulinemic response can be linked to the simultaneous presence of resistant starch, AXs, and β-glucans.
