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gastric phase. Letters a and b indicate the significance of the difference after LSD post-hoc test comparing the two products, first, and the digestive step, secondly.

NOESY JRES

areas Casein Lowpept Casein Lowpept

gastric 3.66±0.30 a,a _3.99±0.24 a,a _3.33±0.05 a,a _3.82±0.05 a,a

duodenal 106.02±0.71 a,b _100.00±5.67 a,b _{107.41±14.73} a,b _100.00±9.03 a,b

These notions are validated by the observation of NMR spectra obtained through the NOESY technique. Visualizing the lowfield region, which shows proton amidic signals (7.3-8.9 ppm), it is possible to gain information regarding water accessibility in exchange sites in peptides. This chemical exchange determines the disappearance, under the water suppressed signal, of amidic signals when peptides are small enough to make all their amidic proton accessible to the solvent. The aromatic region (6.5-7.5 ppm), instead, shows information on non-exchangeable protons belonging to aromatic amino acids, thus approximately 10% of the entire protein composition. The diagnostic power of this two region is clear observing the evolution of spectral profiles during digestion. Initially, the lowfield area is basically flat before gastric digestion, for what concerns casein, and becomes crowded with broad signals after this digestive step, increasing even more the area after the duodenal phase. In Lowpept, this area seems decreasing after the gastric phase, but it shows another great increase after the intestinal digestion. This can be explained by the fact that, after gastric phase, NH2 show that peptones go into solution more slowly than the rate in which peptones become peptides. In the duodenal phase, the solubilization of peptones starts being preponderant again, therefore the net result is a great increase in NH2 signals. To resume the digestive process of these two different samples some spectral parameters where chosen: the ratio between the area of the two food matrices in the regions corresponding to aromatic (AaA) and branched (AaR) aminoacids and the lowfield region where amidic NH2 (ANH) can be found. The

chosen regions where found to be very informative and among the most variable between the digestive steps. By comparison of the areas of these regions it is possible to assess which sample is greatly digested, generating a higher value in the spectral area considered. This can mean that this type of sample could generate a greater quantity of compounds easily accessible for our bodies, such as free aminoacids. Table 5 and 6 show the values of these areas both for casein and Lowpept after gastric and duodenal digestion. The ratio between casein and Lowpept is usually less than 1 in value (Table 7 and 8). Lowpept seems to generate a greater amount of digestive products such

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as peptides and aminoacids.

Table 5 and 6: Mean values and standard deviations for the areas of samples in the different regions. AaA: aromatic

aminoacids region, AaR: branched aminoacids region, ANH: amidic NH2 region. CN stands for casein and LP for LowPept. Letters a and b indicate the significance of the difference after LSD post-hoc test comparing the two products, first, and the digestive step, secondly.

NOESY AaA AaA AaR AaR ANH ANH

Area Casein Lowpept casein Lowpept casein Lowpept

Gastric 0.0451±0.0043a, a _{0.3746±0.0370}b, a _{0.2474±0.0186} a, a _{7.1613±0.1265} a, a _{0.1813±0.0233} a, a _{2.8566±0.0539} a, a

Duodenal 0.0518±0.0021a, b _{0.3904±0.0196} b, b _{0.3046±0.144} a, b _{6.9446±0.3468} a, b _{0.1860±0.0197} a, b _{2.9810±0.1171} a, b

JRES AaA AaA AaR AaR ANH ANH

Area casein Lowpept casein Lowpept casein Lowpept

Gastric 0.0337±0.0050 a, a _{0.0719±0.0098} a, a _{0.6387±0.0829} a, a _{4.7137±0.5895} a, a _{0.2725±0.0077} a, a _{2.4819±0.3280} a, a

Duodenal 0.0497±0.0012 a, b _{0.1033±0.0011}a, b _{0.7411±0.0384} a, b _{5.0639±0.3447} a, b _{0.2358±0.0015} a, b _{2.1098±0.1221} a, b

Table 7: Results from the comparison of spectral areas in the region chosen for aromatic (AaA) and branched (AaR)

aminoacids using NOESY spectra.

Type of AA Phase ppm (start) ppm (end) Casein/Lowpept ratio

AaA Oral 6.78 6.88 0.05 AaR Oral 0.84 0.94 0.11 AaA Gastric 6.78 6.88 0.87 AaR Gastric 0.84 0.94 0.96 AaA Duodenal 6.88 6.92 0.81 AaR Duodenal 0.94 1.06 1.03

Table 8: Results from the comparison of spectral areas in the region chosen for aromatic (AaA) and branched (AaR)

aminoacids using JRES spectra

Type of AA Phase ppm (start) ppm (end) Casein/Lowpept ratio

AaA Oral 6.78 6.88 0.23 AaR Oral 0.84 0.94 0.12 AaA Gastric 6.78 6.88 0.66 AaR Gastric 0.84 0.94 0.69 AaA Duodenal 6.88 6.92 0.86 AaR Duodenal 0.94 1.06 0.93

Comparing the values of areas in these regions, both for branched and aromatic aminoacids, through a two-way Analysis of Variance, the results were the one represented in Tables 5 and 6, the first letter (a or b) shows the difference between casein and Lowpept, whilst the second letter (a or b) shows the difference between gastric and duodenal phase. The difference between digestive steps is always significant, in specific between the duodenal step and the previous two, in

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every region and with every NMR method performed. In addition, NOESY technique, both in the amidic and the aromatic region, shows significant differences among the type of samples, therefore between casein and Lowpept. This is particularly important to understand how real differences between the samples are possible and how this distinction can have an effect on human nutrition. Tyrosine, in effect, lying in the aromatic region, is an aminoacid with many positive functions in the human organisms, such as the synthesis of neurotransmitters like dopamine and norepinephrine [O'Brien et al., 2007], and it has been proven how its assumption is particularly beneficial in conditions of stress or fatigue [Deijen et al., 1999; O'Brien et al., 2007; Mahoney et al., 2007]. In addition to that, other studies have demonstrated how tyrosine can reduce blood pressure in rats [Sved et al., 1979; Yamori et al., 1980]. The branched amino acids analysed, instead, thus leucine, isoleucine and valine, show very similar levels in casein and Lowpept, especially observing NOESY spectra, whilst in JRES spectra, the ratio between the areas of the two samples, shows again a greater value for the Lowpept sample. These amino acids are used for their role in protein synthesis and to produce energy thanks to their aliphatic portion [Brestenský et al., 2015]. They can also contrast fatigue and preserve the immune defense system [Blomstrand, 2006; Calder, 2006], therefore resulting to be very important for our bodies. From these results, it is possible to understand how Lowpept has potentially better nutritional characteristics than casein. Moreover, the fact that a higher quantity of tyrosine is potentially released during digestion from Lowpept further proves the anti-hypertensive properties of this product.

Conclusions

Observing the spectral behaviour of the two samples during digestion, it is possible to see how the area of NMR spectra shows a great increase from the original food product (casein), already in the gastric phase, thanks to protein digestion. Through the study of spectral areas shows it was assessed that the most important step for the further assimilation and absorption of nutrient is the duodenal phase, during which proteins and previously-formed peptides are cut into smaller peptides and free aminoacids. It is evident, therefore, how the digestion of these food matrices is a double step: initially food is digested into big molecules and then, these are split into dimensionally smaller molecules, capable of being absorbed by the intestines. 1H-NMR technique is very useful for the observation of the various steps in protein digestion, allowing to see what happens in every digestive phase and during the entire digestion. As shown, the analysis of determined spectral

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areas, can give many informations on the specific hydrolysis of metabolites of interest, such as protein, in order to understand how a sample can be more digested than another and if molecules of greater nutritional and health interested are formed. In this case, it was demonstrated how Lowpept shows, once again, better nutritional properties, in specific resulting more digestible and assimilable than casein. In this way Lowpept can perform more effectively its anti-hypertensive effect, also thanks to the liberation of a greater quantity of beneficial aminoacids such as tyrosine.

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CHAPTER 7: FOODOMICS IN THE INVESTIGATION OF THE