3.1. TP Chemical Characterization and Fermentation
TPs had very high moisture content that affected their storability (Table 1
). From a chemical point of view, fibres (ADL + hemicellulose + cellulose) were the most abundant fractions; hemicellulose and cellulose came from peel, while ADL (i.e., the more recalcitrant fraction) was attributable, above all, to the lignin, cutin, and suberin of the seed coats [18
]. The remaining fractions, which were described as CS, were composed by oil, protein, sugar, and organic acids, which are the more biodegradable compounds (Table 1
]. Short chain organic acids and ethanol were a relevant fraction of CS of TP for the degradative and fermentation processes in action (Table 1
). Ethanol, lactate, and acetate were the products of the biological metabolism of lactic microorganisms (LAB) present in TP [14
] (Table 2
); pH level (6.86) and remaining fatty acids were typical of aerobic degradative metabolisms [28
]. When fermentation started, the pH dropped immediately to very low values (pH < 4) because of the increase in lactic acid that reached the maximum concentration after 20 days of the process. In fact, lactic acid (pKa
of 3.86) contributed the most to the decline in pH during fermentation, because it is about 10 to 12 times stronger than the other major acids, such as acetic acid (pKa
of 4.75) and propionic acid (pKa
of 4.87). Other LAB metabolites (ethanol and acetate) remained almost constant; on the contrary, no-LAB acids were going to be consumed until only traces remained. A lactic acid/acetic acid ratio was applied as an indicator of LAB fermentation stability [28
]; values of 2–3 meant that stable and optimal conditions were reached from the 20th day of the process; however, the prevalence of LAB was considered as a precautionary measure after 60–100 days when no-LAB metabolites become traces. LAB fermentation is extensively applied as a cheap method in the food preservation industries [29
]. This effect comes about because of the very low pH and anti-microbial compounds production that influences the activity of membrane-bound enzymes and exo-enzymes. In addition, lactic acid is able to enter into the bacteria, lowering cellular pH and killing the microorganisms. Although the complete microorganism elimination occurred for pHs that were lower than 2.5, pHs around 3.5 were effective in eliminating several food-borne pathogens or enteric contaminants after some weeks of treatment [30
The fermentation moderately decreased the TP organic matter content while a great effect occurred on macromolecular composition (Table 1
); as expected, the relative content of CS, the easily biodegradable fraction, increased, and at the same time all fibers decreased. However, quantitative investigation confirmed the CS augmentation and hemicellulose and ADL consumption while no change occurred for cellulose. Pentose sugars that composed hemicellulose were ideal feedstocks for LAB metabolism; ADL had no defined chemical composition, but its LAB consumption was explainable while supposing that microbial activity changed the cell wall structure to make carbohydrates fractions available that were not usable before (e.g., pectin) [31
3.2. TP Polyphenol Composition and Antioxidant Activity
TPC was applied to estimate the fraction of polyphenols in extracts (Table 3
). Total polyphenols belong to a very abundant fraction that is closely-related to the vegetal fiber and a minor one chemically conjugated with small functional groups or free, i.e., the aglycone fraction A-PP [32
]. Polyphenols and fiber had strong ester links that required hard acidic hydrolysis to be broken. On the other hand, soft extraction with water or alcohols was applied to extract the free or weakly linked fractions. The extraction conditions and solvents chosen were additional factors that affected the molecule recovery. Based on previous TP polyphenol characterization, methanol was the best solvent, being chosen to guarantee a very good extraction yield of the more concentrated molecules [33
]. The TPC of TP1 and TP2 were almost similar (Table 3
), which is in agreement with the literature, obtained by applying the same extraction conditions where the range of TPC was into the range 716–3516 mg gallic acid g−1
dry matter [12
]. A more in-depth characterization of the aglycone-polyphenols (A-PP) fraction was carried out in order to better understand which parts of the polyphenols had been extracted, since they were very interesting for their high bioactivity (Table 3
A-PP extracted belonging to the flavonoids and phenolic acids accounted for 70% and 30%, respectively; gallic acid, chlorogenic acid, and cinnamic acid were the more concentrated phenolic acids and naringenin chalcone and naringenin were the most abundant flavonoids (Table 3
). The high naringenin chalcone concentration is due to its accumulation during the naringenin metabolic pathway [34
Based on the HPLC fingerprint, the whole A-PP extracted content was calculated as the sum of phenol acids and flavonoids and compared with the corresponding TPC data (Table 3
). A-PP for both TP1 and TP2 accounted for the greater part of TPC (83% TPC and 96% TPC, respectively, for the TP1 and TP2), thus suggesting that extraction conditions were able to obtain the more active polyphenols fraction.
Bioactivity was firstly measured as antioxidant power (Table 3
) by using IC50
and compared with ascorbic acid (IC50
= 153 μg mL−1
) and Trolox (IC50
= 100 μg mL−1
) known to have high antioxidant capability. The TP1 had the lowest IC50
, while TP2 was similar to Trolox, thus confirming that TP hydrophilic components have good antiradical activity (Table 3
Fermentation has been reported to be able to increase TPC and A-PP for the release from fiber of polyphenol fractions, although the results were not well in agreement based on the biomass, polyphenols typology, and fermentation method adopted [35
In this work, TPC increased 17% and 12.5%, but A-PP significantly decreased during fermentation down to 57 ± 2% as an average of the starting value after 250 days (Table 3
), in agreement with literature [37
No significant A-PP flavonoids qualitative changes occurred between TP and TPF, since all of the molecules become less concentrated with the exception of the quercetin that increased [38
]. Phenolic acids however remained constant and decreased for TP1F and TP2F for the reduction/conservation of most of the molecules with the exception of gallic and cinnamic acids, which increased. Similar behaviors were already described during sourdough LAB fermentation when a global A-PP reduction occurred, while fiber-ferulic acid bound hydrolysis improved the recovery of that phenolic acid [36
TPF showed a significant difference between TPC and A-PP. The explanation is that, although TPC was usually associated with the presence of polyphenols, the Folin–Ciocalteu reagent was not specific for that class of molecules but rather for benzene derived substituents typical not only of polyphenols, but of other several molecules, such as amino acids, sugars, acids, etc. [36
]. The extraction conditions and solvents chosen in addition affected the capability to extract some of the molecules. Since the TPC vs. A-PP difference occurred after fermentation we could suppose that aglycone fractions in the TPC were, in fact, LAB metabolism products. Aromatic organic acids, reducing sugars, and aromatic amino acid are probable constituents with cutin and suberin monomers. The augmentation of the difference after fermentation is, therefore, due to the production of non aglycone molecules. That fraction’s origin was supposed to have originated from polyphenols that were previously fiber-linked and then successively metabolized into de-glucosides, sulfoconjugates, glucoronides, and other forms [40
Bioactivity increased with fermentation for both TPs (+26% and +42% for TP1F and TP2F respectively), nevertheless no significant correlation existed with TPC (r = 0.8, p < 0.2, n = 4). At the same time, A-PP had an opposite trend with respect to IC50, which means that, for TPF, the most part of the antioxidant activity was due to non A-PP.
The explanation becomes more difficult when we consider that polyphenols have different antioxidant power for class, molecules, or derivate metabolites that affect the concentration vs. activity relationship [40
]. The flavonoids were more active in comparison with the phenolic acids and A-PP were more bioactive than bound ones [40
Thus the fraction of molecules defined as (TPC minus A-PP) were correlated with IC50 and a better result was found (r = 0.9, p < 0.1, n = 4) in comparison to that found for TPC vs. IC50. This result indicated that, effectively, TPC-A-PP contained an anti-oxidant fraction, but its presence was not sufficient to totally explain the anti-oxidant activity.
TPC accounted for 10–15% of the methanol-TP extract; therefore, the presence of other antioxidant molecules cannot to be excluded.
Vitamin C, for example, is present in tomato peel, bound to the cell-membrane structure that negatively affected its extractability; recovery was improved by LAB’s capability to break the links, so there was increased vitamin C release after fermentation.
The difficulty found in identifying the molecules that are responsible for the antioxidant effect is typical of what occurs when complex extracts are considered. This is due to the presence of several bioactive substances, since their co-existence generates numerous interactions. Potentiation, antagonism addition, and synergy are known effects that result in the final antioxidant activity deriving from chemical reactions, such as regeneration, spatial distribution, metal chelation, and mutual protection. The presence of pro-oxidant agents, the solubility of antioxidants in reaction media, and the solvent effects might reduce the overall activity [41
The antioxidant bioactivity of TPF does better than that of raw ones thank to the production of fermentation metabolites that increase starting biomass nutritional value and potential health benefits. In addition, LAB fermentation is cheap and guarantees the biomass safety, all positive effects that renewed interest for its application.
3.3. Anti-Inflammatory Properties
Inflammation is a body stress status that is recognized to be the precursor of several diseases. Intestinal cells are able to respond to inflammatory signals by triggering various intracellular signal transduction cascades to control the expression of genes, including cytokines and chemokines, like IL-1, IL-6, IL-8, and TNF-a, due to their high exposure to inflammatory events. The human colon epithelial cell line Caco-2 secretes chemokine IL-8, which directs the migration of leukocytes, monocytes, and macrophages. The ability of polyphenol to influence IL-8 production has been used to evaluate the experimental anti-inflammatory properties.
TP and TPF were tested for their anti-inflammatory capability. The results indicated that all the TP extracts had very high anti-inflammatory effects, being described by a dose-dependent first linear phase followed by a plateau reached in correspondence with the complete inflammation elimination (Figure 1
TPC and A-PP doses were considered with the aim of identifying the TP extract fraction responsible for the effect. Dosing TPC = 15 μg mL−1
TPs were more bioactive than TPF, while the same effect was shown when considering TPC = 25 μg mL−1
, since it corresponded to the plateau phase (Figure 1
a,b). Minimum effective concentration (MEC) i.e., lowest dose that eliminated inflammation completely is a fundamental item of information for applicative purpose; by using the TPC = 15 μg mL−1
dose, it was found that TPF had anti-inflammatory activity of 78% in comparison with TP.
A-PP have been reported to be anti-inflammatory agents, being able to modify the expression of more pro-inflammatory genes, such as multiple cytokines, lipoxygenase, nitric oxide synthases, and cyclooxygenase, with particular reference to the regulation of the expression of NF-kB [42
The doses were recalculated in order to correlate the anti-inflammatory effects of A-PP, resulting in being similar for TP but very different for TPF (Table 4
This dose expression positioned the most part of the samples into the dose-effect range and only TP1_25 and TP2_25 were at plateaus (Figure 1
b). TP and TPF were now aligned to define together a linear straight phase (% reduction= (7.19 ± 0.31) * A-PP, R2
= 0.99, p
< 0.001, n
= 7) (Figure 1
b); this result highlighted that LAB fermentation did not reduce anti-inflammatory capacity, but preserved the starting one and a common MEC of 13.7 μg mL−1
Further investigation led us to conclude that A-PP was responsible for the anti-inflammatory effect and LAB fermentation preserved that bioactivity. With reference to A-PP composition, it is worth noting that TP’s more concentrated flavonoids (naringenin and naringenin chalcone) were more effective than more concentrated phenolic acids (chlorogenic acid and cinnamic) for the capability to interact with different biological targets due to their different structure [43
Naringenin inhibits TNF-α-induced TLR2 expression by inhibiting the nuclear factor-κB (NF-κB) and JNK pathways in adipocyte cells [44
], whereas naringenin chalcone reduced the production of TNF-α and MCP-1 through IκB-α degradation in the RAW 264 macrophages that were stimulated by lipopolysaccharide [45
Among phenolic acids, cinnamic acid and gallic acid inhibited NF-κB activation, in particular phosphorylation of IκB and NF-κB-dependent p65 acetylation, respectively; nevertheless, gallic acid inhibited the activation of COX-2 [43
]. In particular, chlorogenic acid has shown inhibitory activity on the cytokine IL-8 production in the Caco-2 inflamed cells either through directly suppressing the NF-κB activation or indirectly via the inhibition of the upstream signaling pathways [47
The combination of several bioactive substances has been described as enhancing the final anti-inflammatory effect for the capability to act against greater numbers of inflammatory mechanisms at the same time [35
]. However, several chemical interactions affected the bioavailability of the molecules. The flavonoids co-existence improved their chemical stability and solubility, thus positively the bioavailability, but the same molecules competed with phenolic acid for cellular transportation, causing adsorption interference [41
In vegetal extracts, several bioactives co-existed with others, often unknown, thus making it impossible to experimentally understand all of the chemical and biological interactions that contributed to the final bioactivity.
PLS was more reliable than other techniques when identifying relevant variables and their magnitudes of influence, especially in the cases of small sample size and low tolerance [48
]. Coefficient importance was considered a discriminant parameter to select the variables. In this work, PLS was based on the identification of a dose of A-PP molecules vs. anti-inflammatory effect (expressed as IL-8 de-activation) relationship. At the end of several PLS cycles, the best Goodness-of-Fit and Goodness-of-Prediction of the regression model of R2
= 0.95 and R2cv
= 0.62 were reached.
Multiple PLS regression indicated that IL-8 inactivation was well explained by the naringenin chalcone, gallic acid, kaempferol, and apigenin having important coefficient values of 31%, 25%, 24%, 20% respectively. All of the molecules selected represented were the greater part of the A-PP (molecules selected 73 ± 15% of A-PP), and naringenin chalcone and gallic acid together accounted alone for 71 ± 15% of A-PP, therefore contributing the greatest amounts of the molecules’ dose to give bioactivity. On the other hand, from a qualitative point of view, their contribution corresponded to 56% of the importance, while kaempferol and apigenin had greater effects, taking their low concentration into consideration.
PLS selection confirmed the higher bioactivity of flavonoids with respect to phenolic acids (75% and 25% of the importance, respectively) [49
The exclusion of other flavonoids more concentrated than naringenin that demonstrated higher concentration and the selection of kaempferol and apigenin suggested that PLS choice was not based only on the quantitative aspect, but also considered the action mechanism. Kaempferol for example was noted to be very potent phenolic compound for its capacity to affect anti-inflammatory by affecting two different inactivation pathways (inhibited STAT-1 and NF-kB) [50
] on the contrary of the other molecules selected by PLS, where action was addressed to a single biological receptor. Similarly, apigenin has demonstrated strong anti-inflammatory properties via modulation of the gene expression of inflammatory cytokines via acting on the NF-κB and MAPK signaling pathways and through the pro-inflammatory mediators, such as cyclooxygenase, lipoxygenase, and nitric oxide synthases [51