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Four different meat preparation were studied. Each type of meat was either cooked through a traditional method (TR) or with sous-vide (vacuum) cooking (SV). Three samples for each cooking method were analyzed for each meat type, so to have a total of 6 samples for 4 products. Each sample was digested in vitro employing the method described in 13.2. Digested samples were centrifuged and filtered. Before digestion, the protein content of each sample was measured through the Kjeldhal method. Digested samples were analyzed through NMR spectroscopy.

In a 1.5 mL eppendorf, 160 μl of phosphate buffer in deuterated water (D2O) were added to 900 μL

of digested samples. pH was adjusted to 7.0 by means of addition of 1M solutions of either HCl or NaOH. Samples were then centrifuged at 14000 rpm and 4°C for 10 minutes. 800 μl of surnatant were then transferred in NMR tubes for the analysis. NMR spectra were acquired through a 600 MHz spectromoter using an acquisition time of approximately 30 minutes and a NOESY1GPPR sequence. Spectra were referred to the internal standard (TSP) at 0.0 ppm. Peripheral spectral regions and the signal from the solvent were removed from the spectra. First, spectra of the digested samples and the “blank” (only digestive fluids and enzymes) were normalized on the TSP signal to be compared. After a binning procedure the spectrum of the blank was subtracted from all spectra, in order to just observe the signals from the food matrix. The area of the region between 4.15 and 4.66 ppm (“alpha region”) was then calculated, in order to measure the protein release after digestion. To have the real value of the aminoacid in the alpha region, the obtained values were corrected through this equation (Eq. 1):

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where:

R = Area of the Alpha region/Total protein content (measured by Kjeldahl method); N = number of protons in the internal standard (N=9);

C(std) = concentration of the internal standard (TSP); Vs = sample volume;

Vb = buffer volume; Vt = total volume;

PM = average molecular weight of amino acids (PM=110 g/mol).

Means and standard deviations of the obtained values were calculated and compared for each product in the two cooking methods.

Results and discussion

As visible in Figure 7, samples did not show much difference in the amino acid content between the two types of cooking: only slight differences are visible in the average values and when the standard deviation is considered the difference is not statistically significant.

Fig. 7: Mean values with standard deviation for the aminoacid content of digested samples cooked with the traditional

method (blue) or in vacuum (red), measured through NMR spectroscopy and calculated applying equation 1.

The only product showing an increased protein digestibility is pork, where the new cooking method in vacuum proved to increase the product’s protein digestibility. It is clear though that there might exist a particular matrix-related response to the cooking method. Recent studies have confirmed that vacuum cooking is an efficient technique for the improvement of food digestibility. Sangsawad and colleagues [Sangsawad et al., 2016] showed that chicken breast cooked in vacuum at 70°C for 30 minutes had a greater protein digestibility and antioxidant power compared to

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samples cooked at higher temperatures and longer.

In fact, cooking at higher temperatures could cause the formation of indigestible protein aggregates, which could reduce digestion proteolysis and which, in turn, could be fermented by the gut microbiota into potentially damaging molecules (i.e. ammonia) [Oberli et al., 2015]. In this case only one product showed greater protein digestibility, measured as the amount of aminoacids made available through digestion, when the new cooking technique was employed, though this can be due to the fact that a small number of samples was available. For this reason, more research is necessary with greater sample numerosity, in order to further test the possible difference in protein digestibility between the two cooking techniques. With a larger number of samples a lower value of standard deviation can be obtain and significant difference could be found among samples. Moreover, different cooking time and temperature combinations should be evaluated, in order to understand the influence of both factors in the final meat quality and aminoacid realease, as carried out in other studies [Roldan et al., 2014; Oberli et al., 2015]. In fact, both the temperature and the cooking time can impact on meat quality, for example affecting the production of free radicals and thus protein and lipid oxidation. For this reason, it is important to find the best combination of these factors in order to gain greater protein digestibility.

Conclusions

The nutritional value of protein-based food is deeply related to their digestibility and thus to the amount of aminoacid that becomes available during digestion. Vacuum cooking seemed to improve protein digestibility in just one of the four analysed samples (pork meat), showing how the technique can improve the nutritional value of a meat product but the matrix itself can influence the results. Further research with more samples is necessary to prove these findings and gain more robust results. In addition, different combinations of time and temperature of cooking should be tested to understand the impact of each factor and to find the best possible cooking procedure to obtain a higher protein digestibility for each product.

6.4 Digestibility of food supplements – Lowpept Study