*3.8. Antioxidant Performances*

Antioxidant properties of PLOMW were explored by confirming that the grafting reaction has taken place and also to verify whether the reaction conditions were harmful to the active compounds in LOMW. Total phenolic groups, available phenolic acids and antioxidant activity of the conjugate have been reported in Table 5.


**Table 5.** Total polyphenols, phenolic acid contents and antioxidant activity of the conjugate polymer. Data represent mean ± RSD (*n* = 3).

PLOMW = Tara gum grafted with lyophilized olive mill wastewater; APG = Available phenolic groups; PAC = Phenolic acids content; TAC = Total antioxidant activity; DPPH = 2,2-diphenyl-1-picrylhydrazyl radical; ABTS = 2,2-azino-bis(3-ethylbenzothiazolin-6-sulphonic radical; CT = catechin.

Data confirmed that the grafting reaction avoids the loss of the antioxidant activity of LOMW, while BTG did not demonstrate any interference. Specifically, the analyses of the inhibition kinetics against radical species underlined the good performance of PLOMW, both in organic and aqueous environments. However, the response towards the radical ABTS appears almost three times higher, as confirmed by the IC50 values. The collected data clearly indicates as PLOMW is able to guarantee good results and that when introduced into a food, it can impart significant biological properties from a nutritional point of view, producing beneficial effects on the health of consumers.

### *3.9. Pudding Preparation and Evaluation of the Antioxidant Properties*

Gelling properties of the conjugate sugges<sup>t</sup> the employment of this macromolecular system for the preparation of suitable functional foods with high added value. In particular, the PLOMW was used for the production of a fruit-based pudding. The fruit gives numerous benefits to the human body often related to the presence of phenolic compounds. Different studies have correlated the high intake of fruit with the lower incidence of cardiovascular, neurodegenerative and chronic diseases, such as cancer and diabetes [51]. To highlight the effect and benefits derived from using PLOMW in the pudding's preparation, the pear, not particularly rich in antioxidant molecules, was chosen as raw material [52]. Although it can contain in the range 27–41 mg of phenols per 100 g of pulp, the antioxidant profile of pear remains distant from other fruits such as blackberry, raspberry, blueberry, strawberry, cherry and grape [53]. At the same time, this fruit is very digestible and tasty, especially in the preparation of fruit juices for children or hospitalized patients. In this context, puddings based on pear puree were prepared using PLOMW and CTG as gelling agen<sup>t</sup> and their antioxidant capacity was investigated as a function of time. For this purpose, each pudding jar was opened at set time intervals and subjected to an extraction process according to a procedure reported in literature, with some modifications [31]. Antioxidant properties of PPLOMW were analyzed at the opening day (t = 0), after 7, 14 and 28 days. The data of total phenolic groups, phenolic acids and the scavenging properties in aqueous environment are reported in Table 6.

Available phenolic groups (t = 0 days), expressed as mg of CT per gram of pudding, confirm that the addition of the antioxidant polymer PLOMW as gelling agent, significantly increases (almost two times) APG value of PCTG. Similarly, PPOMW at time zero displayed an increased amount of available phenolic acids and more performing scavenging capacity against the hydrophilic radical ABTS. The analysis of the trend of these parameters over 28 days, in terms of APG, highlighted the slight decrease observed for PPOMW (equal to 11% after 28 days). On the contrary, APG decrease equal to 30% after 28 days was recorded with the pudding prepared using CTG. This finding was mainly interesting because it highlights that the employment of the conjugate increased phenolic groups in the functional food and maintained their concentration over time. The same trend was recorded in the content of available phenolic acids, with a decrease after 28 days of 76% for PPLOMW matrix, which increased to 82% in PCTG.

Finally, by recording the inhibition profiles towards the ABTS of the puddings over time, it was observed that the enriched pudding proved to be more effective against this radical specie, compared to the control (Figure 7). IC50 values recorded for the PPLOMW underwent a slight decrease over time (0.0124 mg mL−1, after 28 days). On the contrary, the IC50 values recorded for the pudding based on CTG, underwent a greater decrease (Figure 7).


**Table 6.** Total polyphenols, phenolic acid contents and antioxidant activity of puddings based on PLOMW and CTG. Data represent mean ± RSD (*n* = 3).

PPLOMW = Pudding based on tara gum grafted with lyophilized olive mill wastewater; PCTG = Pudding based on commercial tara gum; APG = Available phenolic groups; PAC = Phenolic acids content; ABTS = 2,2-azinobis(3-ethylbenzothiazolin-6-sulphonic radical; CT = catechin. Different letters express significant differences (*p* < 0.05).

**Figure 7.** IC50 trend as function of the time of PPLOMW and PCTG.

Ultimately, the polymeric conjugate, synthesized using TG and compounds with a polyphenolic structure obtained from waste products of the oil extraction process, inserted in a food matrix such as pudding with pear, has guaranteed it a greater antioxidant capacity and the possibility of keeping it almost unchanged over time.

### *3.10. Rheological Analysis of Puddings*

Rheology is the science that studies the deformation and flow of matter in liquid or solid form, i.e., the response of materials to mechanical stress in terms of viscosity and elasticity [54]. The rheometric measurements of the food matrices PPLOMW and PCTG were carried out by recording the frequency sweeps of the G and G modules as a function of the frequency in the linear viscoelasticity region. The trend of G and G vs. the frequency is also called the mechanical spectrum and allows a quantitative rheological characterization of the materials.

Such a linear viscoelastic behavior has been previously observed in gel systems. All mixtures show a typical gel-like response in which G is higher than G. Higher values of G may reflect the stronger interactions existing among the domains which favor the formation of highly elastic lattices. In order to obtain quantitative information, the weak gel model was applied, which models the system as consisting of connected rheological units. This allowed to determine the values of "A" and "z", where "A" represents the interaction force between the rheological units and "z" the coordination number, that is, the number of rheological units interacting with a reference unit. The "A" and "z" values of the PPLOMW and PCGT food matrices determined by frequency sweep test are shown in Table 7.


**Table 7.** Frequency sweep test for PPLOMW food matrices and PCGT over time.

PPLOMW = Pudding by polymer conjugate; PCTG = pudding by commercial tara gum.

As can be seen from the data reported in Table 7, the values of "A" recorded for PCTG, both at 5 and 25 ◦C increase as functions of time, while the values of "z" are very similar in the first fourteen days and then significantly increased after twenty-eight days. This indicates that initially, the network formed increases the strength between the links and then expands three-dimensionally over time.

Finally, for each sample, temperature ramp tests or time cures were carried out to analyze the behavior of the system as a function of temperature and the results are shown in Figure 8A,B.

**Figure 8.** Time Cure test for the PCGT (**A**) and PPLOMW (**B**) food matrix over time (weeks).

In this type of test, G and G are recorded as a function of temperature at a frequency of 1 Hz within the linear viscoelastic region. The temperature range investigated was 5–40 ◦C, with a scanning speed of 1 ◦C min−1. Figure 8 shows the G comparison (elastic modulus) of the pudding samples prepared with the two polymers investigated (PPLOMW and PCGT) over time. The recorded data indicate that at "time zero" the samples have very different values of elastic modulus (G). In fact, the G values recorded for the PCGT sample are lower than those observed for PPLOMW. However, with the passage of time, the difference between the modules tends to decrease and overlap when both samples reach two weeks of maturation. It is important to note that as the maturation time increases (beyond two weeks), the elastic values of the PPLOMW sample are greater. In fact, the pudding prepared with PPLOMW at "time 4 weeks" shows the highest elastic modulus. This rheological behavior can be attributed to a greater structuring effect induced by the conjugated polymer. Preliminary data sugges<sup>t</sup> that water may play a central role in the structuring through hydrogen bonds, a hypothesis that must necessarily be proved by means of spectroscopic techniques.
