3.1.3. Antimicrobial Activity

Data obtained from measuring the antimicrobial capacity of different extracts are presented in Table 3. The results differed according to the bacterial strain used, *L. monocytogenes* KCTC 3569 CECT 7467 (Gram-positive)*, S. Aureus* ATCC 25923 CECT 435 (Gram-positive) or *E. Coli* O157:H7 ATCC 25922 CECT 434 (Gram-negative). All the extracts showed a lower growth inhibition capacity than cloramphenicol (positive control), a broad spectrum antibiotic.


**Table 3.** Antimicrobial activity of natural extracts measured by the disc difussion method (mm ± SD).

Chloramphenicol: positive control; SD: Standard Deviation. P: Pomegranate extract, RA: Rosemary extract rich in Rosmarinic Acid; NOS: Rosemary extract rich in diterpenes; NOVS: Rosemary extract rich in diterpenes and with lecitin as emulsifier; HYT-L: Hydroxytyrosol extract obtained from olive leaf; HYT-F: Hydroxytyrosol extract obtained from olive fruit.

HYT-L, followed by HYT-F had the highest antimicrobial capacity values against *S. Aureus* ATCC 25923 CECT 435 (gram-positive) growth, because of they prestented 28.1 and 25.2 mm of growth inhibition, respectively. These extracts were followed by RA, P, NOS and NOVS. In this case, clearly, the most active compound was the hydroxytyrosol, as obtained from olive leaves as from olive fruits. In second place was RA, which is the most similar in terms of its molecular structure. Both of them provide an opportunity to study the action mechanism of these compounds in future research. However, the inhibitory capacity of the rest of the compounds was lower and does not allow any structure-activity hypothesis to be proposed.

On the other hand, the gram-positive bacterium *L. monocytogenes* KCTC 3569 CECT 7467 is the most resistant strain to phenolic compounds. In this case, P was the most antimicrobial with 15.3 mm growth inhibition, which corresponds with 44.1% of the positive control, chloramphenicol. This was followed by HYT-L, NOVS, NOS, HYT-F and RA. It can be considered, then, that all the studied compounds showed similar inhibitory activities against this microorganism.

Finally, NOS, was the most antimicrobial extract against the gram-negative bacteria *E. Coli* O157:H7 ATCC 25922 CECT 434, with 20 mm of growth inhibition, 40.6% less than chloramphenicol. This phenolic extact was followed by NOVS, RA, P, HYT-L and HYT-F. In this case, of grea<sup>t</sup> interest and significance is the fact that fat-soluble compounds such as terpenoids had a higher inhibitory capacity against the growth of gram-negative bacteria. This was followed by the rest of extracts, all of which showed similar values of antimicrobial activity, making it difficult to offer any considerations on their structure-activity.

#### *3.2. Influence of Natural Extracts in Oxidative and Microbiological Damage in Fish Products*

#### 3.2.1. Volatile Organic Compounds

Table 4 shows the results obtained from the of GS-MS analysis of the volatile organic compounds: 1-Penten-3-ol, hexanal, 2-nonanone, 1,6-octadien-3-ol, octanal, pentadecane in fish patties. In general, all the volatile compounds analysed increased (*p* < 0.05) from the beginning of storage for all the treatments. These results point to the degradation that is shown in fish patties, that is due to oxidation phenomena, as most straight chain aldehydes are derived from the oxidation of unsaturated fatty acids.




**Table 4.** *Cont.*

Results are expressed as mean ± standard deviation in arbitrary area units (× 106). P: Pomegranate extract, RA: Rosemary extract rich in Rosmarinic Acid; NOS: Rosemary extract rich in diterpenes; NOVS: Rosemary extract rich in diterpenes and with lecitin as emulsifier; HYT-L: Hydroxytyrosol extract obtained from olive leaf; HYT-F: Hydroxytyrosol extract obtained from olive fruit.

Hexanal and 1,6-octadien-3-ol were the dominant aldehyde in the fish patties meat in all the groups. Hexanal values ranged from 0.1 mg/kg, in day 0, to 5.14 mg/kg after 11 days, in control samples. These values are in the same line than those reported by Brunton et al. [28], who found hexanal values of 4.01 l/g in cooked turkey stored for 6 days at 4 ◦C.

Differences in the mean hexanal levels between C and patties with natural extracts were significant (*p* < 0.05) on day 11. On day 11, NOVS showed lower (39%) hexanal values than C, meaning that Rosemary extracts improved lipid stability of the fish patties. In this sense, Shahidi, Yun, and Rubin [29] reported that such an increase in hexanal is a good indicator of lipid oxidation. Indeed, these authors suggested hexanal as a valid indicator of oxidative stability and flavour acceptability in cooked ground meat.

The behaviour of nonanal and 1-penten-3-ol was similar to hexanal, both increasing (*p* < 0.05) during storage and showing significant differences between C and natural extracts samples on day 11. Nonanal is a waxy flavor and descriptors, while 1-penten-3-ol is amongs<sup>t</sup> the compounds responsible for the rancid odour in mayonnaise. Note the absence of significant differences in 2-nonanone and pentadecane on day 11.
