Data represent means and %CV of 6 mice per group; \* *p* < 0.05 versus control.

The IELs subpopulation percentages in jejunum (panel A) and colon (panel B) of mice fed control, wheat bread or wheat–lentil bread diets are presented in Figure 1. Histograms show a significant increase of cytotoxic T cell (CD3+CD8+) percentages in the jejunum of mice fed both types of bread compared to the control diet, whereas the percentage of total T cells (CD3+CD45+) were reduced in mice fed wheat bread compared to control and wheat–lentil bread. In the colon, only a significant increase of B cell (CD19+CD45+) percentages was observed in mice fed wheat–lentil bread. No differences in the percentages of other lymphocyte subpopulations were observed among the groups.

**Figure 1.** Intraepithelial lymphocyte (IEL) subpopulations in the jejunum (**A**) and colon (**B**) of mice fed a control, a wheat bread and a wheat–lentil diet measured by flow cytometry. (The percentage of B and T lymphocytes was calculated on leukocyte (CD45+) gate, whereas the CD4+, CD8+ and CD4+CD8+ subsets, as well as αβ and γδ lymphocytes, were calculated on T lymphocyte (CD3+) gate). Data represent the means ± SD of 6 mice. \* *p* < 0.05 versus control.

Western blot analysis of the phosphorylated form of the p65 subunit of NF-kB in the jejunum and colon of mice did not show any significant difference among groups, indicating that the treatment with wheat and wheat–lentil bread did not induce an inflammatory status in the mice intestine (data not shown).

#### **4. Discussion**

As expected, the proximate composition of the wheat and the wheat–lentil bread mirrored the proximate composition of the flours of origin (see the Materials and Methods section and Table 1). In fact, the wheat–lentil bread contained 30% more proteins than wheat bread, it had an almost double ash content, therefore a higher level of minerals in general, together with an almost double amount of total dietary fibre, especially the insoluble component. Moreover, the lentil–wheat bread contained a lower amount of available carbohydrates than wheat bread.

Besides having a higher protein content, wheat–lentil bread had a more balanced amino acid profile than wheat bread (Table 2). Indeed, the amino acid profiles of wheat and lentils are complementary. For example, lysine is abundant in lentils, whereas sulphur amino acids are present in higher amounts in wheat. Lentil proteins are, in fact, mainly constituted by globulins and albumins [40] and, thus, have a different composition from wheat proteins, which are mainly constituted of prolamins and glutelins. The presence of the lentil flour increases the level of almost all the essential amino acids in bread (Table 2).

The lower amount of available carbohydrates in wheat–lentil bread is due to the fact that lentils contain less starch than wheat (about 40–45%, [18]); it is also reported that legume starch has a higher fraction of amylose than wheat (about 35%, [41]).

Regarding dietary fibre, both wheat and wheat–lentil breads contain only a small amount of the soluble component, around 1%; however, the soluble fibre of lentils is reported to contain beta glucans [18]. Beta-glucans are very interesting from a functional point of view, because they are known to induce a variety of physiological effects with a positive impact on health, acting in particular through immunomodulatory pathways, that can suppress cancer proliferation, lower cholesterol levels and thus reduce the risk for cardiovascular disease [42,43].

The wheat–lentil bread was richer in phenolic substances, in particular those present in the aqueous organic extract, than wheat bread and this is the reason why it also had better antioxidant properties (Table 3). The soluble free phenolics found in the extract come mainly from cellular vacuoles whereas the insoluble phenolics present in the residue are bound to other components mainly fibre. The hydrolysable bound phenols represent the main polyphenol fraction in both bread types (between 73% and 87% of TPC). The literature reports that significant amounts of phenolic compounds remain in the extraction residues, associated with the food matrix [44]. The phenolic molecules most frequently found in cereals are phenolic acids and flavonoids whereas in pulses we also find tannins [45]. Polyphenols in general, both the free and the bound ones, thanks to their antioxidant properties are considered to exert a protective effect on human health [46].

The lignans, secoisolariciresinol and isolariciresinol, were found in both breads (Table 3). However, the wheat–lentil bread not only contained higher amounts of these lignans, but also had additional lignan types and, in particular, lariciresinol and pinoresinol in the following order: Isolariciresinol > lariciresinol > pinoresinol > secoisolariciresinol. These results are in agreement with data on lignan content in legume flours reported by Durazzo et al. [30]. Literature data indicate flaxseed and sesame as major alimentary sources of lignans and rye and lentils as good sources [30,47]. Lignans are a large group of polyphenols of increasing interest because their intake has been related to beneficial health effects, including cancer and cardiovascular disease prevention [48]. In this regard it is interesting to report that in 2012 the research group of During et al. [49] published a paper to report on their investigation of whether plant lignans are taken up by intestinal cells and modulate the intestinal inflammatory response using the Caco-2 cell model. Their findings suggest that plant lignans can be absorbed and metabolised in the small intestine and, among the plants lignans tested, pinoresinol exhibited the strongest anti-inflammatory properties.

The antioxidant power as measured by FRAP was significantly higher in wheat–lentil bread than in wheat bread. The FRAP assay is a quick and sensitive way to measure the antioxidant capacity of biological samples. In both cases, the hydrolysable residue had a higher FRAP value than the aqueous–organic extract thus providing the major contribution to the total antioxidant power (from 69% to 77%); this matches the results of the total polyphenols content. Thus, a bread recipe where about one-quarter of the wheat flour is substituted by red lentil flour more than doubles the antioxidant capacity of bread (Table 3).

Concerning animal experiments, our first consideration was that no significant differences were observed in body weight and food intake among the groups of mice fed the control and the two types of bread diets; this indicates that the different diets had the same palatability for the mice, and that they did not impact on eating behaviour and appetite; and that all the differences observed in our sets of data were due to differences in the composition of the diets.

Analysis of the immune parameters in IELs isolated from jejunum showed an increase of cytotoxic T lymphocytes (CD3+CD8+) percentages in both wheat and wheat–lentil bread-treated animals, as compared to the control. Moreover, total T lymphocytes (CD3+CD45+) were significantly reduced in the wheat bread group and increased in the wheat–lentil bread group, compared to the control (Figure 1A). The IEL subpopulation's analysis in colon showed a significant increase of the B lymphocytes (CD19+CD45+) percentage in lentil bread-treated animals as compared to wheat bread and the control (Figure 1B). In this regard, we could hypothesise a role of the higher amount of β-glucans in the lentil bread while not ignoring that such compounds can increase the percentage of activated B lymphocytes and stimulate immune response [50,51].

We can say that the results of our study indicate a positive effect of wheat–lentil bread supplementation on the intestinal immune system of aged mice, as this supplementation was able to counteract some of the immune alterations typical of the older adults. In fact, aging is characterised by intrinsic changes in hematopoietic precursors that affect their proliferative potential, and this represents a key factor contributing to age-related decline in B- and T-cell production [52]. Thus, the increase of total T lymphocytes indicates a better immune response, and the increase of cytotoxic T lymphocytes suggests an improved capacity to respond to toxic agents and/or pathogens, that is known to be reduced in older adults. We can also hypothesise that the increase of B lymphocytes in the colon indicates a more efficient antibody response. In fact, it is well known that the antibody response is impaired in the older adults [53]. Moreover, it has been largely demonstrated that an antioxidants-containing diet may ameliorate lymphocyte response and protect immune cells from oxidative stress-induced apoptosis [54]. Besides polyphenols in general, the positive effects on the immune system in our specific case could also be ascribed to the significantly higher amount of the lignan isolariciresinol (27 times higher) and the presence of the lignan pinoresinol in wheat–lentil bread compared to wheat bread; these two lignans in particular have been shown to exert immunomodulatory and anti-inflammatory effects [49,55].

No significant differences were observed in the other analysed IELs subpopulations (Figure 1A,B).

Among all the analysed cytokines in serum, only IL-10 was significantly decreased in the wheat and wheat–lentil bread treated animals as compared to the control (Table 6). The role of IL-10 in older adults is controversial; while some studies report that IL-10 increased the inflammatory status, others indicate that this cytokine plays a key role as an anti-inflammatory factor [56,57]. It has also been reported that aging is associated with an increase of IL-10 that, together with other cytokines, could be considered as a marker of frailty [58,59].

#### **5. Conclusions**

It is increasingly coming to general attention that the aging population needs to eat appropriately to prevent and reduce all the health risks associated with this phase of human life. In other words, there is a need for tailored foods for aging people. Enriching staple or widely consumed foods can be a simple strategy to guaranty the intake of key nutrients able to have a beneficial effect on the negative aspects associated with aging such as the decline of the immune function. Based on previous studies done in our laboratory, we identified bread as a target food and red lentil flour as a raw material useful to add functionality to bread. We also identified technological constraints that allowed a maximum addition of 24% lentil flour.

For the purpose of this study, we baked two kinds of bread: A common wheat bread and a wheat–24% lentil flour bread. The chemical analysis of the bread components showed that the wheat–lentil bread had 30% more proteins than wheat bread coupled with a more balanced amino acid composition; it had an almost double mineral as well as total dietary fibre content, especially the insoluble component, double the amount of polyphenols, an interesting lignans content and more than double the antioxidant capacity. Thus, this wheat–lentil bread proved to be nutritionally richer and more functional than common wheat bread.

The in vivo effect of the consumption of wheat–lentil bread versus wheat bread on the immune response was studied by means of a murine model of aged mice. Analysis of the immune parameters in intraepithelial lymphocytes isolated from the mice intestine showed significant differences between the two types of bread indicating a positive effect of the lentil–wheat bread on the intestinal immune system. Cytokines in serum were also analysed. Considering that IL-10 is indicated as a frailty marker, we suppose that wheat and wheat–lentil breads in diets could have a positive effect on inflammatory status and improve the health status of aged mice.

This study clearly demonstrates that this is possible by substituting wheat flour with another suitable flour to manufacture a simple and well-accepted food, such as bread, which shows more functionality and is more tailored for the aging population than traditional, common bread with soft wheat only.

**Author Contributions:** M.C. planned and supervised the research, acquired funds and wrote the final manuscript, V.N. and V.T. performed all the bread analyses but the lignans which were performed by A.D. A.F. and M.R. designed the animal experimental setup and performed the experiments and analysed and interpreted the data. R.R. prepared the experimental diets and took care of the animals.

**Funding:** This research was carried out within the CERSUOM project financed by the Italian Ministry for Agricultural, Alimentary and Forest Policies (MiPAAF).

**Acknowledgments:** Grateful acknowledgements are due to Luigi Bartoli and Francesco Mellara for technical help in the bread preparation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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