ANÁLISIS E INTERPRETACIÓN DEL DIARIO DE CAMPO

Milkshakes samples were enriched with BG-DHA, whilst cookies and pancakes were enriched with AC-DHA. Bioactive enriched foods (BEF) were compared to placebo (PLA) counterparts. Four sampling times were selected: at the beginning of storage (T0) and after 20, 40 and 60 days of storage (T20, T40, T60). The first sampling point was the day when samples arrived in the laboratory. Then, samples were stored at -20°C and sampled after 20, 40 and 60 days from the day of their arrival.

Sample preparation and NMR analysis

For pancakes and milkshakes, at each sampling time a trichloroacetic acid extraction (TCA) was performed on each sample: for this purpose, 5 g of product were added to 15 mL of 7% (w/w) TCA and minced by means of a vertical homogenizer (Ultra-Turrax, Ika®). The resulting product was filtered with filter paper (No. 4) from Whatman (Little Chalfont, Buckinghamshire, HP7 9NA, UK). The pH of a 850 µL aliquot was adjusted to 7.5 using 9 M KOH in an Eppendorf microfuge tube and centrifuged at 14k rpm for 5 min in order to remove the potassium trichloroacetate precipitate. For biscuits samples, a different protocol was adopted, since samples showed an acid hydrolysis during time. The TCA solution was substituted by a phosphate buffer 50 mM, pH 7.4. The so obtained supernatant was stored at −80 °C un l 1_{H-NMR measurements were performed. The samples were}

prepared for NMR analysis by adding 150 μL of a D2O and 10 μL solution of 3-(trimethylsilyl)-

propionic-2,2,3,3-d4 acid sodium salt (TSP) 100 mM to the thawed samples. 1_{H- Samples were}

analysed by applying the NOESY 1D pulse sequence. NMR spectra were recorded at 298 K with a Bruker (Milano, Italy) AVANCE spectrometer, operating at a frequency of 600.13 MHz, equipped with an autosampler with 60 holders. Each spectrum was acquired using 32K data points over a 7211.54 Hz spectral width and adding 256 transients. A recycle delay of 5 s and a 90° pulse of 11.4

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μs were set up. Acquisition time (2.27 s) and recycle delay were adjusted to be 5 times longer than the T1 of the protons under investigation, which was considered not to be longer than 1.4 s. Saturation of residual water signal was achieved by irradiating it during the recycle delay at δ equal to 4.703 ppm. Each spectrum was processed with Top Spin 3.0 (Bruker) by using an automatic command apk0.noe, which performs in one shot the baseline and phase correction and by applying a line-broadening factor of 0.3 Hz. The peaks were assigned by comparing their chemical shift and multiplicity with the literature and by using Chenomx NMR suit 8.1 software.

Pre-processing

After Fourier Transformation, phase and baseline correction, spectra were calibrated with reference to the chemical shift of 0.0 ppm assigned to the internal standard TSP, spectral peripheral regions and the water signal were removed. After this, spectra were normalized referring to the anomeric signals of sugars: sucrose in the case of biscuits, lactose in the case of milkshakes. After normalization and prior to any possible statistical analysis, spectra are binned into intervals of 100 data-points. As a result, the new spectral profile consisted of 655 data-points instead of 65535.

Results and discussion Milkshake

Enriched samples show different signals in milkshake spectra, as shown in the example in Figure 1.

Fig. 1: T0 NOESY1D NMR spectra of enriched (black, red and green) and placebo (blue, light-blue and purple) samples

from the first batch. As visible, enriched spectra show many different signals in comparison to placebo samples and also seem to be less reproducible.

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placebo spectra. These signals are mainly due to organic acids, amino acids (as in Fig. 1) and to aromatic molecules.

Fig. 2: PCA scoreplot calculated for NOESY1D NMR spectra of enriched and placebo milkshake samples from the first

two batches during the 4 different time points.

When samples from different time-points and different batches are evaluated through PCA, it is clear how there are many differences among samples and not mainly caused by the shelf-life itself. As visible in Figure 2, samples from different time-points later than T0 tend to cluster, though with no specific storage-related behavior. Samples from the first batch at T0 seem to differ from any other sample and samples from the second batch at T60 move far from any other type of sample at later storage points and closer to samples at T0 from the first batch.

This behavior might just prove that a lot of difference is there already in the powders at the beginning of storage and these differences can be attenuate by storage time, though this is not always the case. No real differences between placebo and enriched samples is visible by this analysis. In fact, samples seem to differ from each other independently of the enrichment as visible in Figure 3. This shows that the enrichment has no real effect on the matrix behavior during storage.

Biscuits

From the first visual inspection of biscuits spectra, BEF samples had distinctive signals, though just in the aromatic region, whilst other signals just seemed to have a higher intensity for BEF samples, such as in the aliphatic region. Samples from different batches were investigated during the chosen shelf-life to study their behavior.

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