Impact of Lactic Acid Bacteria Fermentation on Phenolic Compounds and Antioxidant Activity of Avocado Leaf Extracts

The growing global consumption of avocados, associated with contents including bioactive compounds with numerous health-promoting properties, is producing a large amount of agro wastes around the world. Different management approaches are available for the recovery of bioactive compounds from wastes as potential ingredients for use in the production of functional foods and nutraceuticals. Lactic acid fermentation can be used to exploit nutritional potential and add value to agro wastes. In this study, fermentations with lactic acid bacteria were carried out in avocado leaves, and the total phenolic content and the antioxidant activity were determined by DPPH and FRAP assays from hydroalcoholic extracts obtained from fermented avocado leaves. Fifteen new phenolic compounds were identified for the first time in avocado leaves by HPLC-ESI-TOF-MS. L. plantarum CECT 748T and P. pentosaceus CECT 4695T showed the highest antioxidant activity. The sum of phenolic compounds was increased by 71, 62, 55 and 21% in fermentations with P. pentosaceus CECT 4695T, L. brevis CECT 5354, P. acidilactici CECT 5765T and L. plantarum CECT 9567, respectively, while it was reduced in the fermentation with L. plantarum 748T by 21% as demonstrated by HPLC-ESI-TOF-MS. Biotransformations induced by bacterial metabolism modified the phenolic compound profile of avocado leaves in a strain-specific-dependent manner. P. pentosaceus CECT 4695T significantly increased kaempferol, P. pentosaceus 4695T, L. brevis 5354 and L. plantarum 9567 increased rutin, and dihydro-p-coumaric acid was increased by the five selected lactic acid bacteria. Total flavonoids were highly increased after fermentations with the five selected lactic acid bacteria but flavonoid glucosides were decreased by L. plantarum 748T, which was related to its higher antioxidant activity. Our results suggest that lactic acid bacteria led the hydrolysis of compounds by enzymatic activity such as glycosidases or decarboxylase and the release of phenolics bound to the plant cell wall, thus improving their bioavailability.


Introduction
Avocado (Persea americana Mill., Lauraceae) is native to southern Mexico, but it is cultivated around the world including in Central and South America, Indonesia, the United States, Australia, South Africa and Spain [1,2], with the Hass variety the most cultivated

Preparation of the Inocula
Strain stocks were cultivated in MRS broth and incubated at 26 • C for 24 h. After growth, the cells were harvested by centrifugation (3000× g for 20 min) to remove the medium and resuspended in 10 mL of sterile saline solution (0.85% NaCl). The bacterial concentration was estimated by turbidimetry and the suspensions were used as inocula for each fermentation. An aliquot was taken from each suspension for counting viable bacteria by enumeration of colonies on MRS agar plates.

Fermentation of Avocado Leaves
Fermentations were carried out as follows: 1 g of the dried ground avocado leaves were submerged in 8 mL of sterile water previously heated to 90 • C. After mixing and cooling, the mixture was supplemented with 1 mL sterile medium containing glucose and yeast extract to obtain a concentration of 0.4% w/v of each. Inocula were added to obtain a concentration between 10 6 and 10 7 cell/mL. CFU/mL counting on MRS agar and pH values were determined at 0, 24, 48, 72 and 96 h of incubation at 26 • C. A control without the addition of LAB strains was included. Two replicates were prepared for each bacteria and control. At the end of incubations, samples and control were stored at −20 • C and then freeze-dried for further determinations.

Polar Compound Extraction
Briefly, 0.2 g of lyophilized avocado leaf fermented powder was dissolved in a 6 mL solution of ethanol/water 80/20, v/v. The mixture was placed in an ultrasonic bath for 15 min, and then it was centrifuged for 10 min at 9000 rpm. The extracting procedure was repeated twice more and all the supernatants were collected, evaporated and reconstituted in 1 mL of methanol/water (50:50, v/v). The final extracts were filtered with regenerated cellulose filters 0.2 µm (Millipore, Bedford, MA, USA) and stored at −18 • C until the analyses.

Determination of Folin-Ciocalteu Reacting Substances
Folin-Ciocalteu spectrophotometric method was used to determine the total Folin-Ciocalteu reacting substances (FCRS) for the first screening in all the fermented avocado leaves [28]. Briefly, 500 µL of the Folin-Ciocalteu reagent was added to 100 µL of the extract. It was added to 6 mL of bi-distilled water and the flask was agitated for a minute. After that, it was added to 2 mL of 15% (w/v) Na 2 CO 3 and filled up to 10 mL with bi-distilled water. The flasks were kept in darkness for 2 h and the measures were carried out at 750 nm and 25 • C with a UV-visible spectrophotometer (Spectrophotometer 300 Array, UV-Vis, single beam, Shimadzu, Duisburg, Germany). Calibration curve was carried out with gallic acid from 1 to 1000 ppm and the equation obtained was y = 0.0012x − 0.0164 (R 2 = 0.9984). Analyses were performed in triplicate and the results are expressed as mg gallic acid equivalents (GAE)/g dry weight (d.w.).

Determination of Antioxidant Activity: DPPH and FRAP Assays
DPPH and FRAP assays were carried out to determine the antioxidant capacity of the avocado leaf fermented by different strains' extracts by the procedures described in previous research [29,30]. Briefly, for the DPPH antioxidant assay, it was mixed with 100 µL of extract with 2.9 mL of the DPPH reagent and the decrease in absorbance was Antioxidants 2023, 12, 298 4 of 17 measured after 30 min at 517 nm. Otherwise, for FRAP it was mixed with 30 µL of extract with 90 µL of distilled water and 900 µL of FRAP reagent, kept at 37 • C for 30 min and the absorbance was measured at 595 nm. The measurements were performed using a UV-visible spectrophotometer (Spectrophotometer 300 Array, UV-Vis, single beam, Shimadzu, Duisburg, Germany). In both assays, Trolox was used as the standard for the calibration curves from 1 to 1000 ppm and the equations obtained were y = 0.0027x + 0.0495 (R 2 = 0.9989) and y = 0.0031x + 0.0016 (R 2 = 0.9934) for the DPPH and FRAP assays, respectively. The analyses were performed in triplicate and the results are expressed in mg of Trolox equivalents (TE)/g of dry weight (d.w.).

Determination of Polar Compounds by HPLC-ESI-TOF-MS
Phenolic compounds present in the fermented and non-fermented avocado leaf extracts were analysed using an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters Corporation, Milford, MA, USA) coupled to an electrospray ionization (ESI) source operating in the negative mode and a mass detector time of flight (TOF) micro mass spectrometer (Waters). The compounds of interest were separated on an ACQUITY UPLC BEH Shield RP18 column (1.7 µm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA) at 40 • C using the conditions and gradient previously stated [31]. H 2 O acidified with 1% of acetic acid and acetonitrile were used as phase A and B, respectively. Analyses were performed in triplicate. The identification of the phenolic compounds was made according to the literature. For ensuring the mass accuracy, the tolerances chosen had a score higher than 90% and error lower than 5 ppm. To quantify the phenolic compounds identified in the avocado leaf extracts, calibration curves were used for vanillic acid (y = 8.1947x + 122.91; R 2 = 0.9976), chlorogenic acid (y = 85.138x + 135.16; R 2 = 0.9978), ferulic acid (y = 16.507x + 92.06; R 2 = 0.9980), quercetin (y = 112.8x + 287.12; R 2 = 0.9957), catechin (y = 41.108x + 335.6; R 2 = 0.9959) and rutin (y = 26.176x + 403.46; R 2 = 0.9924). The results are expressed as µg/g d.w.

Data Processing
The data for the identification of polar compounds in the avocado leaves by HPLC-ESI-TOF-MS were elaborated using MassLynx 4.1 software (Waters Corporation, Milford, MA, USA). Statistical differences (Tukey test) by one-way ANOVA analysis, and Pearson correlations were performed using Statistica 7.0 package (StatSoft, Tulsa, OK, USA). The rest of the statistical analyses were performed using MetaboAnalyst 5.0.

Screening of Lactic Acid Bacteria in Fermentation of Avocado Leaves
As described above, a minimal medium (dextrose plus yeast extract) was added to stimulate the initiation of growth of the inoculated LAB. To check if the treatment with hot water was able to eliminate the microorganisms present in the avocado leaves, viable microorganism counts were performed on MacConkey agar (selective medium for enterobacteria), Tryptic soy agar (TSA, enriched medium for bacteria), and Sabouraud agar (medium for fungi). After incubation of the media, the counting was under the limit of detection of the tests.
As shown in Table S1, avocado leaves did not support the growth of most of the inoculated LAB strains. The number of viable bacteria dropped during the first hours of incubation and continued to decrease gradually throughout the fermentation, with the exception of the two L. plantarum strains, which increased their viable counts. While L. plantarum 748T reached its growth peak at 24 h of incubation (8.44 ± 0.01 log CFU/mL), the exponential phase of growth of L. plantarum 9567 was prolonged until 48 h reaching similar counts (8.41 ± 0.05 log CFU/mL). The growth of the two strains of L. plantarum caused the acidification of the medium ( Figure S1). After 24 h of incubation, the pH dropped from 5.94 to 5.31 with L. plantarum 9567 and to 3.93 with L. plantarum 748T. After 48 h, both pH values were around 4. The pH values for the rest of the strains remained similar to the initial values. Although P. acidilactici strains did not increase their concentrations, they were kept viable during fermentations similar to the initial concentrations.
Poor bacterial growth in avocado leaves can be due to their composition. Some compounds present in fermentation, such as phenolics, can affect the viability and metabolism of LAB [23,32]. Avocado leaves contain glycosides, alkaloids, tannins, saponins, flavonoids, terpenoids and steroids [7][8][9]33] and represent a potential source of antibacterial molecules [5]. Nevertheless, high tolerance to phenolic compounds is found in LAB, especially in members of Lactobacilli [34], which can be isolated from fermented products with a high content of phenolic compounds [35]. L. plantarum has been widely studied for its adaptation to plant habits and capability to metabolise phenolics [24], and it is used as starter in food fermentation.
All the fermented avocado leaves at different hours of incubation were analysed in terms of Folin-Ciocalteu reacting substances (FCRS) and antioxidant activity, and the results are presented in Table 1. As can be seen from the results, the FCRS content ranged from 17.34 to 30.72 mg GAE/g d.w. Otherwise, the antioxidant activity was in the range of 25.56-53.88 and 50.34-96.61 for DPPH and FRAP, respectively. In our study, we applied a heat treatment on the avocado leaves in order to eliminate contaminants that could affect the fermentation process. Thus, avocado leaves were submerged into hot water and then allowed to cool spontaneously. This treatment was carried out both on avocado leaves fermented with lactic bacteria and on the unfermented control. Yamassaki et al. previously reported no decrease in the phenolic content or antioxidant activity when heating avocado leaf hydroalcoholic solutions at 40-100 • C for more than 8 h, and the total phenolic content or antioxidant activity of the extracts did not decrease [36].
An increase in the FCRS content was detected after the fermentation for some microorganisms; however, antioxidant activity by DPPH and FRAP assays was lower after fermentations with LAB strains. For most strains, the highest antioxidant activity was found at 24 and 48 h of fermentation. According to the results, the highest antioxidant activity was obtained after 24 h fermentation with P. acidilactici 5765T in the DPPH assay with 50.01 ± 0.23 mg TE/g d.w. and after 48 h fermentation with L. plantarum 748T with 96.61 ± 1.60 mg TE/g d.w. in FRAP assay. It was previously reported that fermentation of avocado puree with L. plantarum resulted in high levels of total free amino acids and a marked increase in antioxidant activity [37]. However, studies of lactic acid fermentations of avocado leaves are scarce.
Among all the microorganisms tested, the two varieties of L. mesenteroides showed the most minor results at all the different hours evaluated, so they were discarded for the next steps. Regarding the two L. brevis strains, a significant reduction in the FCRS and the antioxidant activity for the 4121T strain was seen; conversely, the 5254 strain produced an increase in FCRS content. Comparing P. acidilactici 5765T and P. acidilactici 98, in the first case, a higher recovery of FCRS was found compared to the control; in the second case, a low amount of FCRS was noticed. When fermenting the avocado leaves with P. pentosaceus strains, the highest results were found at 24 h of fermentation with P. pentosaceus 4695T. Finally, L. plantarum 748T had the best FCRS at 48 h and L. plantarum 9567 at 24 h. Both strains of L. plantarum showed very good performances with significant differences to the control.
The FCRS content was related to the antioxidant activity. A significant positive correlation (p < 0.05) was found between total phenolic content and DPPH (r = 0.7857) and FRAP (r = 0.8069) assays. Likewise, the DPPH assay showed a significant positive correlation with the FRAP assay (r = 0.6107).

Identification of Polar Compounds in Fermented Avocado Leaves by HPLC-ESI-TOF-MS
The selected fermented avocado leaves and a control were characterized by HPLC-ESI-TOF-MS and a total of 48 polar compounds were identified. Among them were seven phenolic acids, thirty-seven flavonoids and four other compounds. They are presented in Table 2 with their experimental and calculated m/z, time (min), error (ppm), score (%), molecular formula and tentative name for each compound. The peaks presented in Table 2 correspond to the numbers shown in Figure 1, which is a representative chromatogram of a fermented avocado leaf. To the best of our knowledge, 15 polar compounds are identified here for the first time in avocado leaves.  Table 2 with their experimental and calculated m/z, time (min), error (ppm), score (%), molecular formula and tentative name for each compound. The peaks presented in Table  2 correspond to the numbers shown in Figure 1, which is a representative chromatogram of a fermented avocado leaf. To the best of our knowledge, 15 polar compounds are identified here for the first time in avocado leaves.   Phenolic acids. Corresponding to peaks 3, 4, 5 and 7 were detected protocatechuic acid-4-glucoside, p-coumaric acid, chlorogenic acid and sinapic acid-C-hexoside in concordance with López-Cobo et al. [38] who previously identified them in avocado by-products. Moreover, at times 6.02 and 7.14, and with the m/z 221 and 165, two coumaric acid derivatives were found named as p-coumaroyl glycolic acid and dihydro-p-coumaric acid, respectively, according to the phenol explorer database [39]. The first one was previously quantified in lentils seeds [40] and the second one in olives [41], and both were found in avocado leaves here for the first time. In addition, a ferulic acid derivative tentatively named as dihydroferulic acid 4-O-glucuronide was detected with the m/z 371, the m/z in source fragment 195 and the predicted molecular formula C 16 H 20 O 10 in agreement with Hu et al. [42] who found it in sweet cherries. Previously, Fan et al. [43] reported a similar compound named ferulic acid 4-O-glucoside in rejected avocados, but it is the first time this compound has been found in avocado leaves.
Flavonoids. Flavan-3-ols are a well-known group of flavonoids usually found in avocado samples, and in this case catechin derivatives were detected corresponding to peaks 8, 11 and 41, named as procyanidin dimer, procyanidin trimer and catechin diglucopyranoside, respectively [13]. Moreover, special attention was paid to the compounds detected at peaks 14 and 17. They were tentatively named as cinchonain-1a-(4beta->8)catechin isomer a and b according to their m/z in source fragments 289 [C 15 H 13 O 6 ] − and 245 [M-3H] 3− (PubChem CID: 442686), and are described in avocado leaves here for the first time. Moreover, the flavonolignan cinchonain was identified at 8.15 min with the m/z 451 [13]. At time 7.57, the compound quercetin (m/z 301) was identified, and a total of 12 quercetin derivatives were detected. With the molecular formula C 27 H 30 O 17 , two isomers named as quercetin-diglucoside isomers a and b were found. Three isomers of quercetin 3-O-arabinosyl-glucoside (a, b and c) were detected with the m/z 595. Corresponding to peaks 23 and 24, two other isomers of a quercetin derivative were identified and named as quercetin-3-glucoside isomers a and b, respectively. In addition, quercetin glucuronide and quercetin-O-deoxyhesoxide were found at times 9.65 and 10.45. All of them were identified in agreement with Castro-López et al. [13] who previously described them in avocado leaves. Also they were found three other quercetin derivatives not reported previously in avocado samples. At 11.02 min the compound named as quercetin 3-O-acetyl-rhamnoside was identified according to Mi et al. [44] who found it in berries. With the m/z 565 and the molecular formula C 25  Four isomers of a luteolin derivative were found with the molecular formula C 26 H 28 O 15 that were called luteolin 7-O-(2"-O-pentosyl)-hexoside isomers a, b, c and d. At 11.81 min with the m/z 563, the compound apigenin-C-hexoside-C-pentoside was identified [13]. Otherwise, kaempferol was detected at 12.3 min with the molecular formula C 15 H 10 O 6 . Its derivatives, kaempferol-O-hexoside isomers a and b, kaempferol 3-O-acetyl-glucoside and kaempferol-O-coumaroyl, were identified corresponding to peaks 30, 34, 44 and 46, respectively [13]. With the m/z 461, the compound kaempferol 3-glucuronide that was previously quantified in strawberry [45] and endive [46] was tentatively identified. Additionally, two isomers (a and b) of kaempferol 3,4 -dixyloside were detected at 10.49 and 10.57 min, respectively, in agreement with Nakane et al. [47] who identified them in leaves of Allium macrostemon. In addition, the compounds detected at peaks 43 and 45 were tentatively named as luteolin 7-[6-O-(2-methylbutyryl)-beta-glucoside] isomers a and b, respectively, according to its fragment 285 that corresponds to C 15 H 10 O 6 (luteolin), and the other to its source fragments 191 and 339 in agreement with Xiong et al. [48] who identified it in sorghum.
Other. Three organic acids and two quinones were detected. Corresponding to peaks 1 and 2, two isomers (a and b) of quinic acid, respectively, were identified [38,49]. Moreover, with the m/z 299, the compounds found at 9.62 and 10.06 min were tentatively identified as two isomers of a trihydroxyanthraquinones named as emodic acid according to its m/z in source fragments 255 [C 14

Quantification of Phenolic Compounds by HPLC-ESI-TOF-MS and Its Biotransformations during Fermentation in Avocado Leaves
The phenolic acids and flavonoids identified in the unfermented and selected fermented avocado leaves were quantified and the results are summarized in Table 3.
Fermentations of avocado leaves with the five selected LAB resulted in modifications in their phenolic profile ( Figure S2). Total phenolic compounds' content was increased by 71, 62, 55 and 21% in fermentations with P. pentosaceus 4695T, L. brevis 5354, P. acidilactici 5765T and L. plantarum 9567, respectively, while it was reduced in the fermentation with L. plantarum 748T by 21% in comparison to the unfermented control. Total phenolic acids were increased by 27, 40 and 43% in P. pentosaceus 4695T, L. brevis 5354 and L. plantarum 9567 fermentations, respectively, but decreased with L. plantarum 748T and P. acidilactici 5765T. With regard to total flavonoids, their content was highly increased by 91, 96 and 75% in P. acidilactici 5765T, P. pentosaceus 4695T and L. brevis 5354 fermentations, respectively.
LAB are able to degrade and biotransform food phenolic compounds by tannase, amylase, esterase, β-glucosidase, phenolic acid decarboxylase (PAD), reductase, or benzyl alcohol dehydrogenase enzymes [14,20]. Hydroxycinnamic acids such as caffeic, p-coumaric or ferulic acids can be reduced into dihydrocaffeic, phloretic or dihydroferulic acids, respectively, or decarboxylated into vinyl derivatives by a phenolic acid decarboxylase enzyme (PAD), and subsequently reduced into ethyl derivatives [50]. While p-coumaric acid was increased in fermentations with P. acidilactici 5765T, P. pentosaceus 4698T and L. brevis 5354, it was reduced after fermentation by L. plantarum 9567 and consumed by L. plantarum 748T (<LOQ). Trans-p-coumaric and cis-ferulic acids were decreased by L. plantarum in cowpeas (Vigna sinensis L.) depending on the isomeric form of the acids; however, spontaneous fermentation increased them [51]. Likewise, L. brevis, L. plantarum and P. pentosaceus were able to metabolize p-coumaric and ferulic acids through decarboxylation [52]. In addition, p-coumaric acid was degraded until p-vinyl-phenol and it reduced derivative dihydro-pcoumaric acid (phloretic acid) by L. plantarum in cherry juice exhibiting a strain-specific metabolism [24]. We found a significant increase in the concentration of dihydro-p-coumaric acid with the five selected strains, especially with P. acidilactici 5765T, P. pentosaceus 4695T and L. brevis 5354, but the decrease in p-coumaric acid in L. plantarum 748T fermentation did not correspond to a higher accumulation of this reduced metabolite compared to the last three bacteria.  Hydroxycinnamic acids are found glycosylated in plants (esterified), covalently attached to the cell wall and as a soluble form in cytoplasm. The breakdown of the ester linkages between polymers release the free phenolic acids [14,53]. Cinnamoyl ester hydrolases, also known as cinnamoyl esterase, catalyse the hydrolysis reaction of hydroxycinnamoyl esters releasing free acids that will be new substrates for phenolic acid decarboxylases PAD [53]. In our study, p-coumaroyl glycolic acid, an ester of p-coumaric acid, was decreased (<LOQ) in the fermentation with L. plantarum 748T. As we mentioned above, this microorganism also consumed p-coumaric acid, suggesting an initial hydrolysis of the ester by a cinnamoyl esterase followed by a decarboxylase activity. Chlorogenic acid, also known as 3-caffeoylquinic acid, is the ester of caffeic and quinic acids, and was significantly decreased in fermentations with all bacteria, especially with P. acidilactici 5765T. Conversely to our results, L. plantarum consumed caffeic, p-coumaric, ferulic, protocatechuic and p-hydroxybenzoic acids except for chlorogenic acid [26]. The ability to hydrolyse cinnamoyl esters of some L. plantarum strains was related to the presence of two esterases with differences in their substrate range: Lp_0796 that hydrolyses esters of caffeic, p-coumaric, ferulic and sinapic acids, while Est_1092 was able to hydrolyse both hydroxycinnamoyl and hydroxybenzoyl esters [54]. This cinnamoyl esterase activity is important in the de-esterification process of dietary fiber in human and ruminal digestion, improving the antioxidant, antiinflammatory and antimicrobial activity of complex dietary compounds [14].
Rutin isomers were increased by P. pentosaceus 4695T, L. brevis 5354 and L. plantarum 9567, while they were reduced by P. acidilactici 5765T (<LOQ). Nevertheless, in fermentation with L. plantarum 748T, rutin isomers a and b were reduced, but isomer c was increased significantly. Kaempferol, kaempferol-3-glucoside, quercetin and quercetin-3-glucoside were released from rutin after fermentation with Aspergillus awamori in Litchi pericarp [55]. Our results showed a significant increase in quercetin-3-glucoside isomers in all fermentations with the exception of L. plantarum 748T. Likewise, a significant increase in kaempferol was found only in the fermentation with P. pentosaceus 4695T, suggesting that this microorganism led to a biotransformation towards this compound. The hydrolysis of rutin to kaempferol-3-rutinoside or quercetin-3-glucoside is catalysed by α-rhamnosidases and further hydrolysed by β-glucosidases to free kaempferol or quercetin [32]. However, free quercetin was found under the limit of quantification (<LOQ) in both fermented and unfermented avocado leaves.
The enzymatic activity of the bacteria can break down vegetable cell walls and release bound phenolics, improving their bioavailability and facilitating their extraction [14,22]. Flavonoids were the main phenolic compound found in avocado leaves and were highly increased after fermentations with LAB. Flavonoids are found predominantly as glycosylated conjugates in plants, mostly as quercetin and kaempferol [56]. With the exception of L. plantarum 748T, the concentrations of flavonoid glucosides such as luteolin-7-O-(2"-Opentosyl)-hexoside isomer a, quercetin-diglucoside and quercetin-3-O-arabinosyl-glucoside were increased compared to the unfermented control. Conversely, luteolin-7-O-(2"-Opentosyl)-hexoside isomer d, quercetin-3-O-arabinosyl-glucoside isomer b and quercetin-3-glucoside isomer b, were reduced significantly by L. plantarum 748T. In soybeans' and mung beans' fermentation with L. plantarum 748T, glycosylated isoflavones were deglycosylated into their respective aglycones increasing their bioavailability [57]. Likewise, cultures of L. plantarum 748T transformed food aryl glycosides: phloridzin, esculin, daidzin and salicin into aglycones with the exception of quercetin glucoside, which remained glycosylated after incubation. The deglycosylation was associated with an increase in the antioxidant activity [58]. Our results showed a decrease in the concentration of quercetin glucoside isomer b but not in isomer a, suggesting a glycosyl hydrolase activity dependent on the isomeric form. In the gut, conjugated glucosides are hydrolysed by the intestinal microbiota to be absorbed into their corresponding aglycone, which show higher activity than their precursor glycosides [23]. β-glycosidase activity is widespread among LAB and have a significant positive impact on fermented products, improving their flavour and fragrance [59]. L. plantarum is commonly found in the human gastrointestinal tract and is used as a starter in the fermentation of dairy products, vegetables and meats [20]. Glycosidase activity of L. plantarum has been associated with an improvement in the bioaccessibility and bioavailability of food phenolic compounds as well as with an increase in their antioxidant activity [58]. Although L. plantarum 748T decreased the total phenolic content of avocado leaves, it showed a significant decrease in aryl-glucosides such as: quercetin-3-glucoside isomer b, protocatechuic acid 4-glucoside, quercetin-3-O-arabinosyl-glucoside isomer b, luteolin 7-O-(2"-O-pentosyl)-hexoside isomer d and kaempferol-O-hexoside isomer b, suggesting a higher glycosidase activity than the rest of the strains. Otherwise, L. plantarum 9567 increased total phenolic compounds by 21% but did not show such marked glucosidase activity as L. plantarum 748T. The higher glycoside deglycosylation shown by L. plantarum 748T, and to a lesser extent by L. plantarum 9567, may be related to the high antioxidant activities found in the DPPH and FRAP assays in comparison with the rest of the bacteria.
A hierarchical clustering heatmap was performed to provide an intuitive visualization of all of the phenolic compounds quantified by HPLC-ESI-TOF-MS in the fermented avocado leaves by the selected strains and a non-fermented control. The features were previously normalized, the distance measure was the Pearson statistical meaning and the clustering method was the average. Therefore, the clustering result for the features (rows) and samples (columns) is shown in Figure 2. Each colour cell on the map corresponds to a concentration value normalized from 2 (intense red) to −2 (intense blue). Moreover, each sample has an associated colour (legend). As can be seen from the figure, the avocado leaf fermented by L. plantarum 748T at 48 h was clustered with the control sample, which shows the minor differences among them in the polyphenol profile, and it had the lowest total phenolic content but with the highest content of chlorogenic acid. Close to them, L. brevis 5354 and P. acidilactici 5765T were also clustered according to their phenolic profile, being the group that showed higher contents As can be seen from the figure, the avocado leaf fermented by L. plantarum 748T at 48 h was clustered with the control sample, which shows the minor differences among them in the polyphenol profile, and it had the lowest total phenolic content but with the highest content of chlorogenic acid. Close to them, L. brevis 5354 and P. acidilactici 5765T were also clustered according to their phenolic profile, being the group that showed higher contents of luteolin and quercetin derivatives. Finally, the group that was clustered furthest from the control was the one composed by P. pentosaceus 4695T and L. plantarum 9567. It seems to be a heterogeneous group in terms of amounts but with a similar profile and proportions between the individual phenolic compounds. Among them, P. pentosaceus 4695T was the strain that led the avocado leaf to release the highest content of coumaric acid derivatives and kaempferol derivatives with the highest total phenolic content. This clustering analysis confirms the strain-specific metabolism of LAB on the phenolic compounds present in avocado leaves, which is dependent on the capability of strains to tolerate and hydrolyse them.

Conclusions
This study allowed us to identify the chemical biotransformations induced by LAB strains in avocado leaves using submerged fermentations. A total of 48 polar compounds were identified by HPLC-ESI-TOF-MS and, to our knowledge, 15 of them were identified for the first time. We found a strain-specific metabolism of the phenolic compounds of avocado leaves, which was dependent on the tolerance of LAB strains to the phenolics' concentration and their capacity to hydrolyse them. Fermentations with P. acidilactici CECT 5765T, P. pentosaceus CECT 4695T, L. plantarum CECT 9567 and L. brevis CECT 5354 led to an increase in the total phenolic content, with the exception of L. plantarum CECT 748T, which decreased it. The phenolic content in fermented leaf extracts was from 21 (L. plantarum CECT 9567) to 71% (P. pentosaceus CECT 4695T), higher than in the control. Briefly, submerged fermentation with lactic acid bacteria can be used in the exploitation and valorisation of avocado agro wastes for the production of enriched phenolic extracts.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox12020298/s1, Figure S1: pH values of lactic acid bacteria cultures during fermentation of avocado leaves; Figure S1: Phenolic profile in fermented and unfermented avocado leaves by HPLC-ESI-TOF-MS; Table S1: Log CFU/mL of lactic acid bacteria in avocado leaves.