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Article

Effect of Lactic Acid Bacteria Fermentation on the Polar Compounds Content with Antioxidant and Antidiabetic Activity of Avocado Seed Extracts

by
María del Carmen Razola-Díaz
1,2,†,
Soumi De Montijo-Prieto
3,†,
María José Aznar-Ramos
1,2,
María Jiménez-Valera
3,
Alfonso Ruiz-Bravo
3,
Vito Verardo
1,2,*,‡ and
Ana María Gómez-Caravaca
2,4,‡
1
Department of Nutrition and Food Science, Campus of Cartuja, University of Granada, 18071 Granada, Spain
2
Biomedical Research Center, Institute of Nutrition and Food Technology ‘José Mataix’, University of Granada, Avda del Conocimiento sn, 18100 Granada, Spain
3
Department of Microbiology, Campus of Cartuja, University of Granada, 18071 Granada, Spain
4
Department of Analytical Chemistry, Campus of Fuentenueva, University of Granada, 18071 Granada, Spain
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
These authors are joint senior authors on this work.
Fermentation 2023, 9(5), 420; https://doi.org/10.3390/fermentation9050420
Submission received: 21 March 2023 / Revised: 18 April 2023 / Accepted: 24 April 2023 / Published: 27 April 2023
(This article belongs to the Special Issue The Role of Antioxidant Compounds in Fermented Foods)
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Abstract

:
Avocado seeds, a common waste in the avocado processing industry, have been found to have several bioactivities, such as anticancer, antimicrobial, hypocholesterolemic, antioxidant, and antidiabetic activities, among others. Nowadays, this wastage is causing an environmental problem, so the use of new technologies to take advantage of it is a novel field of research. In this study, the submerged fermentation by lactic acid bacteria was used as a novel tool for improving the bioactive compound extraction from avocado seeds. Avocado seeds were fermented by different strains, their polar compounds were identified and quantified by HPLC-ESI-TOF-MS, the antioxidant activity was measured by DPPH and FRAP assays, and the antidiabetic activity was analyzed by the alpha-amylase assay. A total of 32 polar compounds were identified, with 13 of them being described in avocado seeds for the first time. Avocado seeds fermented by Pentosaceus acidilactici showed the highest sum of polar compounds (6279.63 ± 67.74 µg/g d.w.), and by extension, of hydroxytyrosol glucoside (2989.76 ± 3.64 µg/g d.w.). Lactiplantibacillus plantarum CECT 9567 showed the highest antioxidant activity measured by both DPPH and FRAP assays (6294.67 ± 19.44 and 6846.91 ± 2.13 µg TE/g d.w., respectively). Furthermore, Lactiplantibacillus plantarum CECT 748T had the highest antidiabetic activity (52.15% ± 0.67% inhibition of alpha-amylase activity), attributable to the polyphenols. According to the results, submerged fermentation by lactic acid bacteria led to an interesting increase of the polar compounds’ extractability of avocado seeds, consequently improving the bioactivities of the extracts, which could then be used for food nutraceutical or cosmetic purposes.

1. Introduction

Avocado (Persea americana Mill., Lauraceae) is a fruit dispersed worldwide in tropical and subtropical regions. There are numerous varieties of avocado according to the growth climate, with Hass being the most well-known and marketed [1]. The avocado processing industry, in the production of guacamole or essential oils, generates a solid waste residue that represents 21–30% of the fruit, consisting of seeds, peels, and exhausted pulp [2]. Usually, those residues are discarded as waste, representing an environmental problem, but they are also a prospective source of bioactive compounds [1]. The avocado seed is composed of the endocarp and the kernel. It has been previously reported to have anticancer activity in oral, breast, and liver cells, attributed to its triterpenoid compounds [3,4]. Additionally, antimicrobial activity of the avocado seed extract against Gram-positive and Gram-negative bacteria has been demonstrated for Salmonella enteritidis, Citrobacter freudii, Pseudomonas aeruginosa, Enterobacter aerogenes, Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli, P. aeruginosa, Mycobacterium intracellulare, and Listeria monocytogenes [5,6,7]. Furthermore, antifungal activity against Candida spp., Cryptococcus neoformans, and Malassezia pachydermatis, and larvicidal activity against Artemia salina and Aedes aegypti, were reported [8]. Its hypocholesterolemic activity was also demonstrated in mice, reducing cholesterol and low-density lipoprotein. In addition, the antioxidant [9] and antidiabetic activity [10] of avocado seed has been previously confirmed.
Lactic acid bacteria (LAB) are a heterogeneous group of Gram-positive food-grade microorganisms, historically used in food preservation. Usually, they are mostly reported to ferment carbohydrates as their main source of carbon to produce lactic acid at the industrial level. They can produce a variety of products, including bacteriocins, vitamins, amines, short-chain fatty acids, and exopolysaccharides, during metabolism [11]. Moreover, there are previous data that report increases in antioxidant and phenolic compounds in food submitted to fermentation by LAB, such as apple juice, mulberry juice, soy milk, and wheat dough. LAB enzymes have the capability to break the cell walls, improving the realization of phenolic acids and flavonoids or converting them into simple forms, in both cases improving their bioavailability [12]. Fermentation with acid lactic bacteria is a low-cost and sustainable technology for maintaining or improving the nutritional quality of food and its sensory properties. In fruits and their byproducts, fermentation can occur spontaneously via the lactic acid microbiota naturally present under adequate conditions. Otherwise, it can also be controlled using lactic acid bacteria starter cultures, aiming at different specific objectives, such as improving the digestibility. The fermentation of avocado seeds by LAB could lead to several benefits, such as improving their bioactivities, a field of research that has not been deeply studied. In avocado leaves, the fermentation by LAB has led to satisfactory results [13]. Recently, Rozan et al. reported a reduction in the anti-nutrients tannins, oxalates, and alkaloids contents of 80.76%, 89.95%, and 70%, respectively, by fermenting avocado seed with LAB [14].
Based on the above and the results obtained in previous research [13], this study aimed to evaluate the effect of submerged fermentation with different LAB on the polar profile of avocado seeds, evaluating the antioxidant and the antidiabetic activities, in comparison to an unfermented avocado seed extract.

2. Materials and Methods

2.1. Chemicals and Samples

Most of the reagents, analytical standards, and the pancreatic alpha-amylase were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were previously reported [13]. Avocado seed byproduct samples of the variety Hass were provided by a local company after the guacamole production, in April 2022. The seeds were freeze-dried, sieved to a 100 µm particle size, and frozen at −18 °C until the analyses.

2.2. Fermentation of Avocado Seeds by Lactic Acid Bacteria

The lactic acid bacteria (LAB) strains used were: Pediococcus acidilactici CECT 5765T, Pediococcus pentosaceus CECT 4695T, Leuconostoc mesenteroides subsp. mesenteroides CECT 219T, Levilactobacillus brevis CECT 5354, Lactiplantibacillus plantarum subsp. plantarum CECT 748T, and Lactiplantibacillus plantarum CECT 9567 (formerly strain C4), and they were obtained and grown as previously reported [13]. Fermentations were carried out according to the methodology described by De Montijo-Prieto et al. [13]. Avocado seeds were submerged in sterile water, previously sterilized at 90 °C, to eliminate the microorganisms previously present. After heat treatment, aliquots were taken to determine viable microorganisms on MacConkey agar (selective medium for enterobacteria, 37 °C), Tryptic soy agar (TSA, enriched medium for bacteria, 37 °C), and Sabouraud agar (medium for fungi, 25 °C). After incubations of the media, the number of viable microorganisms was confirmed to be below the detection limit of the tests. The quantification of the phenolic compounds was carried out at selected incubation times. Thus, P. acidilactici CECT 5765T, P. pentosaceus CECT 923, Le. brevis CECT 5354, and La. plantarum CECT 9567 were analyzed at 24 h of incubation, and Leu. mesenteroides CECT 219T and La. plantarum CECT 748T at 48 h of incubation.

2.3. Determination of Polar Compounds by HPLC-ESI-TOF-MS

The extraction and analysis of polar compounds in the fermented and non-fermented avocado seeds were carried out following the procedure previously reported by De Montijo-Prieto et al. [13]. Analyses were performed in triplicate. The identification of the phenolic compounds was performed according to the literature. For ensuring the mass accuracy, the tolerances chosen had a score higher than 90% and an error lower than 5 ppm. To quantify the phenolic compounds identified in the avocado seed extracts, calibration curves were used for vanillic acid (y = 4.1347x + 393.22; R2 = 0.982), chlorogenic acid (y = 101.17x − 683.25; R2 = 0.9933), ferulic acid (y = 10.502x + 353.77; R2 = 0.9964), quercetin (y = 288.76x + 2462.1; R2 = 0.9673), catechin (y = 315.87x − 415.59; R2 = 0.9950), and rutin (y = 860.4x − 256.21; R2 = 0.9986). The results are expressed as µg/g d.w.

2.4. Determination of Antioxidant Activity: DPPH and FRAP Assays

Avocado seeds fermented by different LAB were analyzed with DPPH and FRAP assays to determine the antioxidant activity via the procedures described in previous research [15]. The analyses were performed in triplicate and the results are expressed in mg of Trolox equivalents (TE)/g of dry weight (d.w.).

2.5. Determination of Antidiabetic Activity: Alpha-Amylase Assay

The antidiabetic activity of the extracts was performed in vitro by the alpha-amylase assay according to previous research [16,17]. Briefly, 100 µL of fermented or non-fermented methanolic extract of avocado seed (20 mg/mL) was added to 100 µL of porcine pancreatic alpha-amylase (≥2 µd/mL) and pre-incubated during 5 min at 37 °C. Then, 200 µL of 0.2% starch solution in 20 mM of sodium phosphate buffer (pH 6.7) was added and incubated again for 5 min at 37 °C. The reaction was stopped with 1 mL of dinitrosalicylic acid (DNS) color reagent and kept in a boiling bath for 5 min. Once the tubes had cooled on ice until reaching room temperature, they were added to 6 mL of distilled water. The absorbance was measured at 540 nm in a UV-visible spectrophotometer (Spectrophotometer 300 Array, UV-Vis, single-beam, Shimadzu, Duisburg, Germany). The pharmacological inhibitor, acarbose, was included as a positive control at a concentration of 20 mg/mL. Analyses were performed in triplicate. The percentage of inhibition of the alpha-amylase activity was calculated as follows (Equation (1)):
%   I n h i b i t i o n = ( 100 − A b s o r b a n c e   c o n t r o l − A b s o r b a n c e   s a m p l e A b s o r b a n c e   c o n t r o l ) × 100

2.6. Data Processing

MassLynx 4.1 software (Waters Corporation, Milford, MA, USA) was used for elaborating the HPLC-ESI-TOF-MS results. The Statistica 7.0 package (StatSoft, Tulsa, OK, USA) was used for obtaining statistical differences (Tukey’s test) by one-way ANOVA, and Pearson’ correlations were performed. The rest of the statistical analyses were performed using MetaboAnalyst 5.0 (Xia Lab, McGill, Montréal, QC, Canada).

3. Results and Discussion

3.1. Identification of Polar Compounds in Fermented Avocado Seeds by HPLC-ESI-TOF-MS

The avocado seeds fermented by the selected strains were analyzed by HPLC-ESI-TOF-MS, and a total of 32 polar compounds were identified. Among them were six phenolic acids, eighteen flavonoids, five terpenoid derivatives, one tyrosol derivative, and other organic compounds. They are presented in Table 1, along with their experimental and calculated m/z, time (min), error (ppm), score (%), molecular formula, m/z main in source fragments, and the tentative name for each compound. The peaks presented in Table 1 correspond to the numbers shown in Figure 1, which is a representative chromatogram of a fermented avocado seed. To the best of our knowledge, 13 polar compounds were identified here for the first time in avocado seeds.
Corresponding to peaks 3 and 4, two isomers of quinic acid were detected, named as isomers a and b, respectively. Some quinic acid derivatives were detected conjugated with other phenolic acids. Caffeoylquinic acid isomers a and b were detected at 2.57 and 4.58 min (peaks 6 and 10) with the m/z in source fragments 179 (C9H7O4)− of caffeic acid and 191 (C7H11O6)− of quinic acid, respectively. Besides, four isomers of coumaroylquinic acid, named as a, b, c, and d (peaks 7, 8, 11, and 15, respectively), were identified according to its m/z in source fragments 163 (C9H7O3)− and 191 (C7H11O6)−, belonging to coumaric and quinic acids, respectively [18].
With the m/z 625, two quercetin-diglucoside isomers named a and b were detected according to its m/z in source fragment 300 (C15H8O7)2− (peaks 23 and 24), that corresponds to quercetin-7-olate, a conjugate base of quercetin arising from selective deprotonation of the 7-hydroxy group [18].
The organic compound perseitol, also known as D-glycero-D-galacto-heptitol, a sugar alcohol, was identified in avocado seeds at 0.33 min (peak 2) according to its m/z in source fragments 59 (C2H3O2)−, 89 (C3H5O3)−, and 101 (C4H5O3)− [18].
The iridoid penstemide was identified corresponding to peak 9 with the m/z of 443 and the fragmentation according to López-Cobo et al. [18].
A phenethyl alcohol derivative, hydroxytyrosol glucoside (peak 5), was also found in fermented avocado seeds with the molecular formula C14H20O8, the m/z 315, and the m/z in source fragment 153 that corresponds to hydroxytyrosol [18].
At 7.20 min, the compound found with the m/z 441 was named as hydroxyabscisic acid beta-D-glucoside, a derivative of abscisic acid, an isoprenoid plant hormone [18].
Catechin, procyanidin dimer, trimer, and tetramer were detected at 5.22, 5.62, 7.31, and 6.98, respectively, in accordance with other authors [18].
In addition, other compounds were identified here for the first time in avocado seed. With the m/z 343, peak 1 was named as the galloyl ester 5-O-galloyl-1,4-galactarolactone according to the PUBCHEM database (PubChem CID: 59235421). The same molecule was also identified, but it was not quantified in Phyllanthus emblica L. fruit [19].
Tuberonic acid beta-D-glucoside (peak 12 at 5.02 min) was previously described by some authors in rice [20] and leaves of tobacco [21].
Corresponding to peak 14, a new flavonoid was detected with the m/z 449 and the predicted formula C21H22O11. It was named as eriodictyol 7-O-glucoside, in concordance with its main m/z in source fragment 287 (C15H11O6)−, that is eriodyctiol and the phenol explorer database [22]. This compound has been previously identified and quantified in other food, such as almonds [23].
With the m/z 439, three flavonoid isomers were identified and called triacetoxy-methoxy-methylflavone isomer a, b, and c, according to the PUBCHEM database (PubChem CID: 129815511). Its main m/z in source fragment 287 (C15H11O6)− suggests that it could also be an eriodictyol derivative, but it has not been previously described.
In addition, other flavonoid derivatives were identified. Three isomers (a, b, and c) of a luteolin/kaempferol derivative were identified according to m/z 463 (peaks 18, 19, and 25) and the m/z in source fragments 285 (C15H9O6)− and 125 (C6H9O5)−, that correspond to luteolin and galloyl glucose with the loss of the carboxyl group, compounds that were previously identified and quantified in avocado seed [24]. Another luteolin derivative (II) was detected at 10.66, 10.70, 11.19, and 11.32 min, named as isomers a, b, c, and d, respectively. Their m/z in source fragment 285 (C15H9O6)− is in agreement as they correspond to luteolin or kaempferol [24].

3.2. Lactic Acid Bacteria Growth in Avocado Seed

Avocado seed mixtures were supplemented with glucose and yeast extract to encourage the growth of LAB strains. As shown in Table 2, the number of viable bacteria in the avocado seed gradually dropped during incubations, until concentrations between 3.88 ± 0.09 and 4.17 ± 0.03 log CFU/mL at 48 h of incubation. Nevertheless, La. plantarum CECT 9567 slightly decreased from 7.31 ± 0.02 to 6.71 ± 0.03 log CFU/mL at 24 and 48 h of incubation, respectively. As can be seen in Table 2, all LAB had a negative significant (p < 0.05) correlation between the CFU/mL and the time (r = −0.9964). Therefore, when increasing the time, a lower CFU/mL was found in all cases. Despite being part of the LAB metabolism, phenolic compounds are sometimes toxic for them, causing decreases in the growth. The specific pathway of polyphenols that produces bacterial inhibition is still unclear, but changes in the membrane fatty acid composition in La. plantarum were previously reported [25]. Therefore, plant-associated LAB have developed metabolic routes for degradation of those polyphenols by enzymes into less toxic compounds. Among the enzymes described in LAB strains capable of degrading or bio-transforming phenolic compounds are tannase, amylase, esterase, β-glucosidase, phenolic acid decarboxylase (PAD), reductase, and benzyl alcohol dehydrogenase enzymes [26]. Tannase enzymes can hydrolyze tannins and gallic acid esters. Hydroxycinnamic acids such as caffeic, p-coumaric, and ferulic acids can be bio-transformed into dihydrocaffeic, phloretic, or dihydroferulic acids, respectively, by reductase enzymes, or they can be decarboxylated into vinyl derivatives, such as vinyl phenol and vinyl guaiacol, by PAD enzymes. Subsequently, those vinyl derivatives can be reduced into ethyl derivatives [27]. Glucosidase enzymes release phenolic compounds by hydrolysis of glycosylated precursors, including flavonoid glucosides [28]. The tolerance to phenolic compounds depends on the LAB specie. A positive significant correlation was found between the content of quinic acid isomers a and b and perseitol, with the Log10 CFU/mL of all lactic acid bacteria (r = 0.6313, 0.6895, and 0.9481, respectively). In these cases, those compounds could have inhibited the growth of LAB during the incubation time or were highly available during fermentation of the avocado seed and were accessible for the bacteria to be used or bio-transformed into other compounds as part of their metabolism. In contrast, quercetin-diglucoside isomer a and luteolin derivative II isomer b showed a negative significant correlation with Log10 CFU/mL (r = −0.4490 and −0.5098, respectively). This means that those compounds were formed because of bio-transformations from precursor compounds and that were not toxic for LAB. Regarding P. acidilactici CECT 5765 T, it showed a significant (p < 0.05) positive correlation of caffeoylquinic acid isomers a and b and the sum of procyanidins, with Log10 CFU/mL (r = 0.9999, 0.9999, and 0.9624, respectively). Additionally, hydroxytyrosol glucoside and perseitol had the same tendency (p < 0.05, r = 0.9999, in both cases). P. pentosaceus CECT 4695 T, Le. brevis CECT 5354, and La. plantarum CECT 9567 had similar tendencies. In the case of Leu. mesenteroides CECT 219 T, the phenolic acid precursor quinic acid isomers a and b showed a significant positive correlation with Log10 CFU/mL (r = 0.9278 and 0.8662, respectively). Furthermore, perseitol, catechin, and hydrotytyrosol glucoside showed a significant positive correlation with the Log10 CFU/mL (r = 0.9902, 0.8845, and 0.9954, respectively). In contrast, caffeoylquinic acid isomers and coumaroylquinic acid isomers had significant negative correlations with Log10 CFU/mL (r from −0.7225 to −0.9995). For procyanidin dimer, trimer, and tetramer, the opposite correlation was found with Log10 CFU/mL compared to catechin. In the case of La. plantarum CECT 748 T, only quinic acid isomers a and b, perseitol, and hydroxytyrosol glucoside showed significant positive correlations with Log10 CFU/mL (r > 0.9622), and negative correlations were found for the rest of the compounds. Previously, the inhibition of the growth of La. plantarum with the presence of caffeic acid and its derivatives has been reported by other authors [25]. Others reported inhibition of La. plantarum by p-coumaric and ferulic acids present in wines [29]. Additionally, the inhibition of other LAB by hydroxycinnamic acids has been reported [30], or inhibition of other Gram-positive bacteria such as Bacillus cereus and Enterococcus faecalis by caffeoylquinic acid [31]. Furthermore, phenolic compounds found in olive products, such as oleuropein, tyrosol, synapic acid, and syringic acid, showed inhibitory activity against La. plantarum strains [32]. Otherwise, there are no previous references of catechin inhibiting the growth of LAB [33]. The use of an avocado seed hydrolysate-based medium to support biomass growth and lactic acid production by Lactobacillus sp. has been previously reported [34]. As previously reported [35], avocado seed is a well-known source of carbohydrates (65%), with perseitol and D-mannuheptulose being the sugar compounds that stand out the most. Therefore, according to the results, the content of perseitol was reduced with the increase of Log10 CFU/mL, which indicates that its content directly affects the growth of the LAB tested.
Taking everything into account, in order to compare between the LAB strains, the extracts fermented by Leu. mesenteroides CECT 219 T and La. plantarum CECT 748 T at 48 h and the rest at 24 h were selected. This is also supported by previous research [13].
The pH throughout the fermentations decreased from 5.96 ± 0.02 (initial) to values between 4.5 and 5.2 at 48 h of incubation.

3.3. Quantification of Phenolic Compounds by HPLC-ESI-TOF-MS and Its Bio-Transformations during Fermentation in Avocado Seeds

The phenolic and other polar compounds identified in fermented and non-fermented avocado seeds are summarized in Table 3.
As can be seen in Table 3, the sum of polar compounds ranged between 4013 and 6280 µg/g d.w. in the avocado seed fermented by LAB. Increments of 26% in avocado seed fermented by P. acidilactici and 4.3% by Leu. mesenteroides were observed compared to the control, but decreases were found in avocado seeds fermented by other LAB.
The sum of procyanidins decreased in the case of fermentation with P. acidilactici, P. pentosaceus, and Le. brevis, and did not show significant differences by Leu. mesenteroides or both strains of La. plantarum compared to the control. However, no significant increases were seen. There were no significant differences detected for the flavonoids eriodictyol 7-O-glucoside, 2-phenylethyl beta-primeveroside, and quercetin-diglucoside isomer a. Regarding luteolin derivatives, avocado seed fermented by La. plantarum CECT 748T stood out, with the highest content. Otherwise, some decreases were detected for quercetin diglucoside isomer b and the three isomers of triacetoxy-methoxy-methylflavone in the avocado seeds fermented by Le. brevis, P. pentosaceus, and P. acidilactici. Furthermore, perseitol significantly decreased in all cases; meanwhile, penstemide did not change compared to the control avocado seed. Most likely, perseitol was converted into D-mannoheptulose [36] by some of the LAB that have aldolase enzymes and consumed as a nutrient for their metabolism [37].
As shown in Table 3, the sum of phenolic acids and precursors increased by 32.21% and 3.74% after fermentations with P. acidilactici CECT 5765T and Leu. mesenteroides CECT 219T, respectively, but decreased with the other bacteria. Nevertheless, lactic acid bacteria metabolism led to interesting bio-transformations in the avocado seed compounds. Hydroxytyrosol glucoside, an important derivative of hydroxytyrosol in olive fruits [38,39], was significantly increased in fermentations with P. acidilactici CECT 5765T, P. pentosaceus CECT 923, Leu. mesenteroides CECT 219T, and Le. brevis CECT 5354. These increases can be explained by the fact that lactic acid bacteria can release phenolic compounds from plant matrices through their hydrolytic enzymes, such as amylase, xylanase, glucosidase, cellulase, tannase, esterase, or chitinase. These enzymes can disrupt the cell wall, where phenolic acids are found linked to lignin, cellulose, hemicellulose, or structural proteins through ether bonds via the hydroxyl group in aromatic rings, or they form esters via the carboxylic group with proteins and carbohydrates [40]. Thus, these enzymes play a key role in disintegrating plant cell wall matrices and increasing the bioavailability of phenolic compounds, which can be used as a new substrate for other bacterial enzymes or can be free for their extraction and recovery as added-value bioactive compounds. On the other hand, the fermentation of avocado seed with the two La. plantarum strains significantly decreased the hydroxytyrosol glucoside content. This decrease could be a result of the hydrolysis via the glucosidase activity of both strains to release hydroxytyrosol and glucose. Glycosidase activities are widespread in lactic acid bacteria; among them, La. plantarum glycosidases have been especially studied [41]. These enzymes have been reported to release a large number of secondary metabolites from their glycosylated precursors, increasing their antioxidant activity and improving the flavor and aroma of fermented foods [28]. In addition to olive fruit, hydroxytyrosol and hydroxytyrosol glucoside have previously been found in avocado seed [18]. They have shown antioxidant, antibacterial, antifungal, anti-inflammatory, and antitumoral activities in both in vitro and in vivo assays [42,43,44]. In our previous research with avocado leaves [13], fermentation with both La. plantarum strains decreased the concentration of flavonoid glucosides and other glycosylated phenolic compounds, and in the case of La. plantarum CECT 748T, it led to a high antioxidant activity.
Regarding chlorogenic acids found in avocado seeds, a significant increase was found in caffeoylquinic acid in fermentation with La. plantarum CECT 9567 and in coumaroylquinic acid isomers a and c with P. acidilactici CECT 5765T, P. pentosaceus CECT 923, Leu. mesenteroides CECT 219T, and Le. brevis CECT 5354 fermentations. This group of phenolic compounds are produced in plants by esterification of the quinic acid and the hydroxycinnamic acids, including caffeic acid, p-coumaric acid, and ferulic acid [45]. They are widely distributed in plant-based foods, such as coffee, tea, and wine, as well as in avocado seed [46]. The most abundant compound is 5-caffeoylquinic acid. Their occurrence in plants has been associated with lignin biosynthesis, as an indirect defense agent, making tissues more resistant to pathogen attacks and inhibiting fungal toxins, and as a protective agent against oxidative damage caused by exposition to salinity and heat [45]. They have shown antioxidant, antimicrobial, antiviral, and anti-inflammatory activity, as well as risk reduction of developing type 2 diabetes and cardiovascular diseases [47]. Despite the antimicrobial activity, some microorganisms are resistant to chlorogenic acids because of their ability to degrade such compounds [47]. In this study, caffeoylquinic acid isomers a and b and coumaroylquinic acid isomers b and d decreased in fermentations with P. acidilactici CECT 5765T, P. pentosaceus CECT 923, Leu. mesenteroides CECT 219T, and Le. brevis CECT 5354, which could be due to the esterase activity of the strains. In human digestion, when chlorogenic acids reach the colon, the microbiota hydrolyses them, releasing hydroxycinnamic acid and quinic acid [48]. A significant increase in the concentration of quinic acid isomers a and b was found when avocado seeds were fermented with Le. brevis CECT 5354 and La. plantarum CECT 9567, indicating possible hydrolysis of hydroxycinnamic acids. The hydrolysis of these acyl-quinic acids is carried out by cinnamoyl esterases, which have been studied in Lactobacillus species, including La. plantarum [49] and L. johnsonii [50], as well as in Escherichia coli, Bifidobacterium lactis, and Lactobacillus gasseri [51]. Although hydroxycinnamic acids are efficiently absorbed after hydrolysis, intact chlorogenic acids are less well-absorbed only in the small intestine [52]. The hydrolysis of dietary phenolics by fermentation before intake can increase the bioavailability of bioactive compounds in case of dysbiosis of the fermentative microbiota [53].

3.4. Antioxidant Activity in Fermented Avocado Seeds

The antioxidant activity of the LAB-fermented avocado seed extracts was analyzed by DPPH and FRAP assays compared to non-fermented extract of avocado seeds (control), and the results are shown in Figure 2. A significant (p < 0.05) positive correlation (r = 0.8884) was found between the antioxidant activity measured by both methods, DPPH and FRAP.
As can be seen in Figure 2, the antioxidant activity obtained for the non-fermented and fermented avocado seed extracts ranged 3803–6295 and 3714–6847 µg TE/g d.w. for DPPH and FRAP assays, respectively. In both cases, the lowest result corresponded to P. pentosaceus CECT 4695T, and the highest to La. plantarum CECT 9567, with significant differences with respect to the control. For the FRAP assay, La. plantarum CECT 748T and Leu. mesenteroides CECT 219T also showed significant increases compared to the control. In the DPPH results, no significant reduction in the antioxidant activity was found for Leu. mesenteroides CECT 219T. Although avocado seed fermented by P. acidilactici CECT 5765T showed the highest content in the sum of polar compounds and, by extension, in the sum of phenolic acids and precursors, it showed a significant decrease in its antioxidant activity compared to the control. In contrast, those that had increases in their antioxidant activity did not show significant increases in the sum of polar compounds. This phenomenon was previously noticed by Li et al. [54] in apple juice fermented by La. plantarum ATCC14917. They reported a significant decrease in total phenolic and total flavonoid compounds during fermentation, while the antioxidant activity increased over time. They attributed this to the increase of 5-O-caffeoylquinic acid, quercetin, and phloretin [54]. Results found here agree that La. plantarum CECT 9567, compared to the control, had significant increments of quinic acid isomer a, and caffeoylquinic acid isomers a and b. This was also statistically confirmed. According to the Pearson’s correlation test, the antioxidant activity in the fermented avocado seed can be attributed to the polyphenol content. Furthermore, the sum of procyanidins showed a significant (p < 0.05) strong correlation for both methods, DPPH (r = 0.9406) and FRAP (r = 0.8865). Besides, caffeoylquinic acid isomers a and b and coumaroylquinic acid isomers b and d, penstemide, eriodyctiol 7-O-glucoside, quercetin-diglucoside isomers a and b, and triacetoxy-methoxy-methylflavone isomers a, b, and c also had significant (p < 0.05) strong correlations with the antioxidant activity, as measured by both methods. Previously, Rozan et al. also reported an increment in the DPPH antioxidant activity in Hass avocado kernel flour fermented by La. plantarum [14].

3.5. Antidiabetic Activity in Fermented Avocado Seeds

The capacity of inhibiting alpha-amylase was analyzed in the LAB-fermented avocado seed compared to the non-fermented extract of the avocado seed (control). All were tested at 20 mg of avocado/mL, and the results are shown in Figure 3.
As can be seen in Figure 3, all the LAB tested presented a % of inhibition higher than 25%. La. plantarum CECT 748T had the highest % of inhibition; meanwhile, P. acidilactici CECT 5765T had the lowest. La. plantarum CECT 9567 did not show significant differences with the unfermented avocado seed, but the rest had significant decreases in the capacity of inhibiting alpha-amylase activity. Ojo et al. reported values similar to those obtained here, with a % of inhibition ≈20% of alpha-amylase at ≈20 mg/mL of avocado seed extract [10]. Moreover, Abd Elkader et al. reported values of the same range of magnitude with avocado leaves and fruits [55]. The Pearson correlation test revealed a significant positive correlation between the antidiabetic activity and the compounds caffeoylquinic acid (isomers a and b, r = 0.7786 and 0.6163, respectively), penstemide (r = 0.5154), procyanidin trimer (r = 0.6112) and tetramer (r = 0.6881), quercetin-diglucoside isomers a (r = 0.7749) and b (r = 0.6540), eriodyctiol 7-O-glucoside (r = 0.7022), triacetoxy-methoxy-methylflavone isomers b (r = 0.5741) and c (r = 0.5421), and luteolin/kaempferol derivative isomers a–d (r = 0.5929–0.7970). Moreover, a positive correlation (r = 0.7568) with total flavan-3-ols content was also found. Younis et al. [56] studied the antidiabetic activity of seven varieties of avocado seed by the alpha-amylase method and reported that it could be attributed to its content of polyunsaturated fatty acids, as well as several polyphenolic compounds, such as eriodictyol-7-oneohesperidoside, 4-caffeoylquinic acid, and linoleic acid ethyl ester [56]. Furthermore, the inhibition of alpha-amylase activity showed a significant positive correlation with the antioxidant activity measured by DPPH (r = 0.6376) and FRAP assays (r = 0.5503). This makes sense as most of the compounds correlated with the antioxidant activity also showed correlation with the antidiabetic activity, and by extension, flavonoids. The differentiation compounds between both activities were luteolin derivative isomers. In fact, flavonoids are reported by other authors to have antidiabetic activity [57].

3.6. Clustering Heatmap

A hierarchical clustering heatmap (Figure 4) was performed to provide an intuitive visualization of all the polar compounds quantified by HPLC-ESI-TOF-MS and the antioxidant (DPPH and FRAP) and antidiabetic (alpha-amylase) assays carried out in the fermented avocado seeds by the selected strains, and the non-fermented control.
As can be seen from Figure 4, avocado seed fermented by La. plantarum CECT 9567 was clustered with the control samples, showing minor differences. Close to them were the avocado seeds fermented by La. plantarum CECT 748T and Leu. mesenteroides CECT 219T. Although fermentation of the avocado seed with La. plantarum CECT 9567 showed a phenolic profile similar to that of the unfermented seed, key bio-transformations carried out by this strain can be visualized in the figure by color changes. These include increased concentrations of quinic acid and chlorogenic acids, such as some isomers of caffeoylquinic acid, as well as decreased concentrations of hydroxytyrosol-glucoside, by enzymatic hydrolysis for the release of free hydroxytyrosol and glucose. Furthermore, Figure 4 highlights the antioxidant activity by the FRAP assay found in the fermentation with this strain. Another clear group was formed by the rest of the LAB-fermented avocado seeds. In a nutshell, this clustering confirms all the results discussed previously, and shows the differences in the metabolism of the tested LAB on the polar compounds present in avocado seeds.

4. Conclusions

In this research, the chemical bio-transformations and changes in antioxidant and antidiabetic activity induced by LAB strains were studied in avocado seeds using submerged fermentations. A total of 32 polar compounds were identified by HPLC-ESI-TOF-MS and, to our knowledge, 13 of them were tentatively identified for the first time. It was found that among all the compounds, caffeoylquinic and coumaroylquinic acids’ isomers could have inhibited the growth of L. mesenteroides. No effect was found for procyanidins in LAB growth. Quinic acid and perseitol seemed to be the most available compounds to be bio-transformed by LAB or consumed as part of their metabolism. Avocado seeds fermented by P. acidilactici showed the highest sum of polar compounds, and by extension, of hydroxytyrosol glucoside, quantified by HPCL-ESI-TOF-MS. Among all the tested LAB, La. plantarum CECT 9567 showed the highest significant increase in the antioxidant activity, measured by both DPPH and FRAP, mainly attributable to its content in hydroxycinnamic acids, procyanidins, and flavonoids. Furthermore, La. plantarum CECT 748T had the highest alpha-amylase inhibitory activity, attributable to the polyphenols. In conclusion, submerged fermentation by lactic acid bacteria can be used in the exploitation and valorization of avocado seed waste for its use in food formulations or the production of enriched extracts.

Author Contributions

Conceptualization, V.V. and A.M.G.-C.; methodology, M.J.-V., A.R.-B., V.V. and A.M.G.-C.; formal analysis, S.D.M.-P. and M.d.C.R.-D.; investigation, M.d.C.R.-D., S.D.M.-P. and M.J.A.-R.; resources, V.V., A.M.G.-C., M.J.-V. and A.R.-B.; data curation, A.R.-B. and V.V.; writing—original draft preparation, M.d.C.R.-D. and S.D.M.-P.; writing—review and editing, A.R.-B., V.V. and A.M.G.-C.; supervision, M.J.-V., A.R.-B., V.V. and A.M.G.-C.; project administration, V.V. and A.M.G.-C.; funding acquisition, V.V. and A.M.G.-C. All authors have read and agreed to the published version of the manuscript.

Funding

This study is part of the national project RTI2018-099835-A-I00, financed by MCIN/AEI/10.13039/501100011033/FEDER, “Una manera de hacer Europa”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Padilla-Camberos, E.; Martínez-Velázquez, M.; Flores-Fernández, J.M.; Villanueva-Rodríguez, S. Acute Toxicity and Genotoxic Activity of Avocado Seed Extract (Persea americana Mill., c.v. Hass). Sci. World J. 2013, 2013, 245828. [Google Scholar] [CrossRef] [PubMed]
  2. Araújo, R.G.; Rodriguez-Jasso, R.M.; Ruiz, H.A.; Pintado, M.M.E.; Aguilar, C.N. Avocado by-products: Nutritional and functional properties. Trends Food Sci. Technol. 2018, 80, 51–60. [Google Scholar] [CrossRef]
  3. D’Ambrosio, S.M.; Han, C.; Pan, L.; Douglas Kinghorn, A.; Ding, H.; D’Ambrosio, S.M.; Han, C.; Pan, L.; Douglas Kinghorn, A.; Ding, H. Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway. Biochem. Biophys. Res. Commun. 2011, 409, 465–469. [Google Scholar] [CrossRef] [PubMed]
  4. Abubakar, A.N.F.; Achmadi, S.S.; Suparto, I.H. Triterpenoid of avocado (Persea americana) seed and its cytotoxic activity toward breast MCF-7 and liver HepG2 cancer cells. Asian Pac. J. Trop. Biomed. 2017, 7, 397–400. [Google Scholar] [CrossRef]
  5. Raymond Chia, T.W.; Dykes, G.A. Antimicrobial activity of crude epicarp and seed extracts from mature avocado fruit (Persea americana) of three cultivars. Pharm. Biol. 2010, 48, 753–756. [Google Scholar] [CrossRef]
  6. Falodun, A.; Erharuyi, O.; Imieje, V.; Ahomafor, J.; Akunyuli, C.; Jacobs, M.; Khan, S.; Hamann, M.T.; Langer, P.; Biotechnol Author manuscript, N.J. In vitro evaluation of aliphatic fatty alcohol metabolites of Perseaamericana seed as potential antimalarial and antimicrobial agents. Niger. J. Biotechnol. 2014, 27, 1–7. [Google Scholar] [CrossRef]
  7. Salinas-Salazar, C.; Hernández-Brenes, C.; Rodríguez-Sánchez, D.G.; Castillo, E.C.; Navarro-Silva, J.M.; Pacheco, A. Inhibitory Activity of Avocado Seed Fatty Acid Derivatives (Acetogenins) Against Listeria Monocytogenes. J. Food Sci. 2017, 82, 134–144. [Google Scholar] [CrossRef]
  8. Leite, J.J.G.; Brito, É.H.S.; Cordeiro, R.A.; Brilhante, R.S.N.; Sidrim, J.J.C.; Bertini, L.M.; De Morais, S.M.; Rocha, M.F.G. Chemical composition, toxicity and larvicidal and antifungal activities of Persea americana (avocado) seed extracts. Rev. Soc. Bras. Med. Trop. 2009, 42, 110–113. [Google Scholar] [CrossRef]
  9. Soledad, C.P.T.; Paola, H.C.; Carlos Enrique, O.V.; Israel, R.L.I.; GuadalupeVirginia, N.M.; Raúl, Á.S. Avocado seeds (Persea americana cv. Criollo sp.): Lipophilic compounds profile and biological activities. Saudi J. Biol. Sci. 2021, 28, 3384–3390. [Google Scholar] [CrossRef]
  10. Ojo, O.A.; Amanze, J.C.; Oni, A.I.; Grant, S.; Iyobhebhe, M.; Elebiyo, T.C.; Rotimi, D.; Asogwa, N.T.; Oyinloye, B.E.; Ajiboye, B.O.; et al. Antidiabetic activity of avocado seeds (Persea americana Mill.) in diabetic rats via activation of PI3K/AKT signaling pathway. Sci. Rep. 2022, 12, 2919. [Google Scholar] [CrossRef]
  11. Wang, Y.; Wu, J.; Lv, M.; Shao, Z.; Hungwe, M.; Wang, J.; Bai, X.; Xie, J.; Wang, Y.; Geng, W. Metabolism Characteristics of Lactic Acid Bacteria and the Expanding Applications in Food Industry. Front. Bioeng. Biotechnol. 2021, 9, 612285. [Google Scholar] [CrossRef]
  12. Fadlillah, H.N.; Nuraida, L.; Sitanggang, A.B.; Palupi, N.S. Production of Antioxidants Through Lactic Acid Fermentation: Current Developments and Outlook. Ann. Univ. Dunarea Jos Galati Fascicle VI Food Technol. 2021, 45, 203–228. [Google Scholar] [CrossRef]
  13. De Montijo-Prieto, S.; Razola-Díaz, M.D.C.; Barbieri, F.; Tabanelli, G.; Gardini, F.; Jiménez-Valera, M.; Ruiz-Bravo, A.; Verardo, V.; Gómez-Caravaca, A.M. Impact of Lactic Acid Bacteria Fermentation on Phenolic Compounds and Antioxidant Activity of Avocado Leaf Extracts. Antioxidants 2023, 12, 298. [Google Scholar] [CrossRef]
  14. Rozan, M.; Alamri, E.; Bayomy, H. Fermented Hass avocado kernel: Nutritional properties and use in the manufacture of biscuits. Saudi J. Biol. Sci. 2022, 29, 103295. [Google Scholar] [CrossRef] [PubMed]
  15. Razola-Díaz, M.d.C.; Aznar-Ramos, M.J.; Guerra-Hernández, E.J.; García-Villanova, B.; Gómez-Caravaca, A.M.; Verardo, V. Establishment of a Sonotrode Ultrasound-Assisted Extraction of Phenolic Compounds from Apple Pomace. Foods 2022, 11, 3809. [Google Scholar] [CrossRef] [PubMed]
  16. Poovitha, S.; Parani, M. In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (Momordica charantia L.). BMC Complement. Altern. Med. 2016, 16 (Suppl. S1), 185. [Google Scholar] [CrossRef] [PubMed]
  17. Lordan, S.; Smyth, T.J.; Soler-Vila, A.; Stanton, C.; Paul Ross, R. The α-amylase and α-glucosidase inhibitory effects of Irish seaweed extracts. Food Chem. 2013, 141, 2170–2176. [Google Scholar] [CrossRef]
  18. López-Cobo, A.; Gómez-Caravaca, A.M.; Pasini, F.; Caboni, M.F.; Segura-Carretero, A.; Fernández-Gutiérrez, A. HPLC-DAD-ESI-QTOF-MS and HPLC-FLD-MS as valuable tools for the determination of phenolic and other polar compounds in the edible part and by-products of avocado. LWT—Food Sci. Technol. 2016, 73, 505–513. [Google Scholar] [CrossRef]
  19. Zhang, Y.J.; Tanaka, T.; Yang, C.R.; Kouno, I. New phenolic constituents from the fruit juice of Phyllanthus emblica. Chem. Pharm. Bull. 2001, 49, 537–540. [Google Scholar] [CrossRef]
  20. Wakuta, S.; Hamada, S.; Ito, H.; Matsuura, H.; Nabeta, K.; Matsui, H. Identification of a beta-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.). Phytochemistry 2010, 71, 1280–1288. [Google Scholar] [CrossRef] [PubMed]
  21. Sato, C.; Seto, Y.; Nabeta, K.; Matsuura, H. Kinetics of the accumulation of jasmonic acid and its derivatives in systemic leaves of tobacco (Nicotiana tabacum cv. Xanthi nc) and translocation of deuterium-labeled jasmonic acid from the wounding site to the systemic site. Biosci. Biotechnol. Biochem. 2009, 73, 1962–1970. [Google Scholar] [CrossRef] [PubMed]
  22. Rothwell, J.A.; Perez-Jimenez, J.; Neveu, V.; Medina-Remón, A.; M’hiri, N.; García-Lobato, P.; Manach, C.; Knox, C.; Eisner, R.; Wishart, D.S.; et al. Phenol-Explorer 3.0: A major update of the Phenol-Explorer database to incorporate data on the effects of food processing on polyphenol content. Database 2013, 2013, bat070. [Google Scholar] [CrossRef] [PubMed]
  23. Timón, M.; Andrés, A.I.; Sorrentino, L.; Cardenia, V.; Petrón, M.J. Effect of Phenolic Compounds from Almond Skins Obtained by Water Extraction on Pork Patty Shelf Life. Antioxidants 2022, 11, 2175. [Google Scholar] [CrossRef] [PubMed]
  24. Lyu, X.; Agar, O.T.; Barrow, C.J.; Dunshea, F.R.; Suleria, H.A.R. Phenolic Compounds Profiling and Their Antioxidant Capacity in the Peel, Pulp, and Seed of Australian Grown Avocado. Antioxidants 2023, 12, 185. [Google Scholar] [CrossRef]
  25. Rozès, N.; Peres, C. Effects of phenolic compounds on the growth and the fatty acid composition of Lactobacillus plantarum. Appl. Microbiol. Biotechnol. 1998, 49, 108–111. [Google Scholar] [CrossRef]
  26. Rodríguez, H.; Curiel, J.A.; Landete, J.M.; de las Rivas, B.; de Felipe, F.L.; Gómez-Cordovés, C.; Mancheño, J.M.; Muñoz, R. Food phenolics and lactic acid bacteria. Int. J. Food Microbiol. 2009, 132, 79–90. [Google Scholar] [CrossRef]
  27. Filannino, P.; Gobbetti, M.; De Angelis, M.; Di Cagno, R. Hydroxycinnamic acids used as external acceptors of electrons: An energetic advantage for strictly heterofermentative lactic acid bacteria. Appl. Environ. Microbiol. 2014, 80, 7574–7582. [Google Scholar] [CrossRef]
  28. Michlmayr, H.; Kneifel, W. β-Glucosidase activities of lactic acid bacteria: Mechanisms, impact on fermented food and human health. FEMS Microbiol. Lett. 2014, 352, 1–10. [Google Scholar] [CrossRef]
  29. Landete, J.M.; Rodríguez, H.; De Las Rivas, B.; Muñoz, R. High-Added-Value Antioxidants Obtained from the Degradation of Wine Phenolics by Lactobacillus plantarum. J. Food Prot. 2007, 70, 2670–2675. [Google Scholar] [CrossRef]
  30. Filannino, P.; Cardinali, G.; Rizzello, C.G.; Buchin, S.; De Angelis, M.; Gobbetti, M.; Di Cagno, R. Metabolic responses of Lactobacillus plantarum strains during fermentation and storage of vegetable and fruit juices. Appl. Environ. Microbiol. 2014, 80, 2206–2215. [Google Scholar] [CrossRef]
  31. Fiamegos, Y.C.; Kastritis, P.L.; Exarchou, V.; Han, H.; Bonvin, A.M.J.J.; Vervoort, J.; Lewis, K.; Hamblin, M.R.; Tegos, G.P. Antimicrobial and efflux pump inhibitory activity of caffeoylquinic acids from Artemisia absinthium against gram-positive pathogenic bacteria. PLoS ONE 2011, 6, e18127. [Google Scholar] [CrossRef] [PubMed]
  32. Landete, J.M.; Curiel, J.A.; Rodríguez, H.; de las Rivas, B.; Muñoz, R. Study of the inhibitory activity of phenolic compounds found in olive products and their degradation by Lactobacillus plantarum strains. Food Chem. 2008, 107, 320–326. [Google Scholar] [CrossRef]
  33. Figueiredo, A.R.; Campos, F.; de Freitas, V.; Hogg, T.; Couto, J.A. Effect of phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii. Food Microbiol. 2008, 25, 105–112. [Google Scholar] [CrossRef]
  34. Espinel-Ríos, S.; Palmerín-Carreño, D.M.; Hernández-Orihuela, A.L.; Martínez-Antonio, A. A plackett-burman design for substituting mrs medium components with avocado seed hydrolysate for growth and lactic acid production by lactobacillus sp. Rev. Mex. Ing. Quim. 2019, 18, 131–141. [Google Scholar] [CrossRef]
  35. Bangar, S.P.; Dunno, K.; Dhull, S.B.; Kumar Siroha, A.; Changan, S.; Maqsood, S.; Rusu, A.V. Avocado seed discoveries: Chemical composition, biological properties, and industrial food applications. Food Chem. X 2022, 16, 100507. [Google Scholar] [CrossRef]
  36. Tesfay, S.Z.; Bertling, I.; Bower, J.P. D-mannoheptulose and perseitol in “Hass” avocado: Metabolism in seed and mesocarp tissue. S. Afr. J. Bot. 2012, 79, 159–165. [Google Scholar] [CrossRef]
  37. London, J. Variations in the Quaternary Structure of Three Lactic Acid Bacteria Aldolases. J. Biol. Chem. 1974, 249, 7977–7983. [Google Scholar] [CrossRef]
  38. Romero, C.; Brenes, M.; García, P.; Garrido, A. Hydroxytyrosol 4-β-D-glucoside, an important phenolic compound in olive fruits and derived products. J. Agric. Food Chem. 2002, 50, 3835–3839. [Google Scholar] [CrossRef]
  39. Fernández-Prior, Á.; Bermúdez-Oria, A.; Fernández-Bolaños, J.; Espejo-Calvo, J.A.; López-Maestro, F.; Rodríguez-Gutiérrez, G. Evolution of Hydroxytyrosol, Hydroxytyrosol 4-β-d-Glucoside, 3,4-Dihydroxyphenylglycol and Tyrosol in Olive Oil Solid Waste or “Alperujo”. Molecules 2022, 27, 8380. [Google Scholar] [CrossRef]
  40. Muñoz, R.; de las Rivas, B.; López de Felipe, F.; Reverón, I.; Santamaría, L.; Esteban-Torres, M.; Curiel, J.A.; Rodríguez, H.; Landete, J.M. Biotransformation of Phenolics by Lactobacillus plantarum in Fermented Foods. In Fermented Foods in Health and Disease Prevention; Elsevier Inc.: Amsterdam, The Netherlands, 2017; pp. 63–83. [Google Scholar] [CrossRef]
  41. Landete, J.M.; Curiel, J.A.; Rodríguez, H.; de las Rivas, B.; Muñoz, R. Aryl glycosidases from Lactobacillus plantarum increase antioxidant activity of phenolic compounds. J. Funct. Foods 2014, 7, 322–329. [Google Scholar] [CrossRef]
  42. Fernandez-Bolanos, J.; Lopez, O.; Fernandez-Bolanos, J.; Rodriguez-Gutierrez, G. Hydroxytyrosol and Derivatives: Isolation, Synthesis, and Biological Properties. Curr. Org. Chem. 2008, 12, 442–463. [Google Scholar] [CrossRef]
  43. Bertelli, M.; Kiani, A.K.; Paolacci, S.; Manara, E.; Kurti, D.; Dhuli, K.; Bushati, V.; Miertus, J.; Pangallo, D.; Baglivo, M.; et al. Hydroxytyrosol: A natural compound with promising pharmacological activities. J. Biotechnol. 2020, 309, 29–33. [Google Scholar] [CrossRef] [PubMed]
  44. Caballero-Guerrero, B.; Garrido-Fernández, A.; Fermoso, F.G.; Rodríguez-Gutierrez, G.; Fernández-Prior, M.Á.; Reinhard, C.; Nyström, L.; Benítez-Cabello, A.; Arroyo-López, F.N. Antimicrobial effects of treated olive mill waste on foodborne pathogens. LWT 2022, 164, 113628. [Google Scholar] [CrossRef]
  45. Clifford, M.N.; Jaganath, I.B.; Ludwig, I.A.; Crozier, A. Chlorogenic acids and the acyl-quinic acids: Discovery, biosynthesis, bioavailability and bioactivity. Nat. Prod. Rep. 2017, 34, 1391–1421. [Google Scholar] [CrossRef]
  46. Tremocoldi, M.A.; Rosalen, P.L.; Franchin, M.; Massarioli, A.P.; Denny, C.; Daiuto, É.R.; Paschoal, J.A.R.; Melo, P.S.; De Alencar, S.M. Exploration of avocado by-products as natural sources of bioactive compounds. PLoS ONE 2018, 13, e0192577. [Google Scholar] [CrossRef]
  47. Valanciene, E.; Malys, N. Advances in Production of Hydroxycinnamoyl-Quinic Acids: From Natural Sources to Biotechnology. Antioxidants 2022, 11, 2427. [Google Scholar] [CrossRef] [PubMed]
  48. Naranjo Pinta, M.; Montoliu, I.; Aura, A.M.; Seppänen-Laakso, T.; Barron, D.; Moco, S. In Vitro Gut Metabolism of [U-13C]-Quinic Acid, The Other Hydrolysis Product of Chlorogenic Acid. Mol. Nutr. Food Res. 2018, 62, 1800396. [Google Scholar] [CrossRef]
  49. Esteban-Torres, M.; Landete, J.M.; Reverón, I.; Santamaría, L.; de las Rivas, B.; Muñoz, R. A Lactobacillus plantarum esterase active on a broad range of phenolic esters. Appl. Environ. Microbiol. 2015, 81, 3235–3242. [Google Scholar] [CrossRef] [PubMed]
  50. Kin, K.L.; Lorca, G.L.; Gonzalez, C.F. Biochemical properties of two cinnamoyl esterases purified from a Lactobacillus johnsonii strain isolated from stool samples of diabetes-resistant rats. Appl. Environ. Microbiol. 2009, 75, 5018–5024. [Google Scholar] [CrossRef]
  51. Couteau, D.; McCartney, A.L.; Gibson, G.R.; Williamson, G. Isolation and characterization of human colonic bacteria able to hydrolyse chlorogenic acid. J. Appl. Microbiol. 2001, 90, 873–881. [Google Scholar] [CrossRef]
  52. Clifford, M.N.; Kerimi, A.; Williamson, G. Bioavailability and metabolism of chlorogenic acids (acyl-quinic acids) in humans. Compr. Rev. Food Sci. Food Saf. 2020, 19, 1299–1352. [Google Scholar] [CrossRef] [PubMed]
  53. Filannino, P.; Di Cagno, R.; Gobbetti, M. Metabolic and functional paths of lactic acid bacteria in plant foods: Get out of the labyrinth. Curr. Opin. Biotechnol. 2018, 49, 64–72. [Google Scholar] [CrossRef] [PubMed]
  54. Li, Z.; Teng, J.; Lyu, Y.; Hu, X.; Zhao, Y.; Wang, M. Enhanced Antioxidant Activity for Apple Juice Fermented with Lactobacillus plantarum ATCC14917. Molecules 2019, 24, 51. [Google Scholar] [CrossRef] [PubMed]
  55. Abd Elkader, A.M.; Labib, S.; Taha, T.F.; Althobaiti, F.; Aldhahrani, A.; Salem, H.M.; Saad, A.; Ibrahim, F.M. Phytogenic compounds from avocado (Persea americana L.) extracts; antioxidant activity, amylase inhibitory activity, therapeutic potential of type 2 diabetes. Saudi J. Biol. Sci. 2022, 29, 1428–1433. [Google Scholar] [CrossRef]
  56. Younis, I.Y.; Khattab, A.R.; Selim, N.M.; Sobeh, M.; Elhawary, S.S.; Bishbishy, M.H.E. Metabolomics-based profiling of 4 avocado varieties using HPLC–MS/MS and GC/MS and evaluation of their antidiabetic activity. Sci. Rep. 2022, 12, 4966. [Google Scholar] [CrossRef]
  57. Al-Ishaq, R.K.; Abotaleb, M.; Kubatka, P.; Kajo, K.; Büsselberg, D. Flavonoids and their anti-diabetic effects: Cellular mechanisms and effects to improve blood sugar levels. Biomolecules 2019, 9, 430. [Google Scholar] [CrossRef]
Fermentation 09 00420 g001 550
Figure 1. Base peak chromatogram of the polar compounds identified in the fermented avocado seed. Numbers 1–36 correspond to the peaks in Table 2.
Figure 1. Base peak chromatogram of the polar compounds identified in the fermented avocado seed. Numbers 1–36 correspond to the peaks in Table 2.
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Figure 2. Antioxidant activity of avocado seed fermented by LAB strains and a control measured by DPPH and FRAP assays. Different letters (a–g) for each method indicate significant differences.
Figure 2. Antioxidant activity of avocado seed fermented by LAB strains and a control measured by DPPH and FRAP assays. Different letters (a–g) for each method indicate significant differences.
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Figure 3. Inhibition of alpha-amylase activity of fermented avocado seed by different strains and non-fermented avocado seed. Different letters (a–d) indicate significant differences.
Figure 3. Inhibition of alpha-amylase activity of fermented avocado seed by different strains and non-fermented avocado seed. Different letters (a–d) indicate significant differences.
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Figure 4. Clustering heatmap of the avocado seeds fermented by LAB and the control according to the analyses performed.
Figure 4. Clustering heatmap of the avocado seeds fermented by LAB and the control according to the analyses performed.
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Table
Table 1. Polar compounds identified by HPLC-ESI-TOF-MS in fermented and non-fermented avocado seeds.
Table 1. Polar compounds identified by HPLC-ESI-TOF-MS in fermented and non-fermented avocado seeds.
No.Time (min)m/z Experimentalm/z CalculatedError (ppm)Score (%)Molecular Formulam/z in Source FragmentsCompound
10.26343.0304343.03010.994.75C13H12O11201.02265-O-Galloyl-1,4-galactarolactone
20.33211.0813211.0823−4.791.29C7H16O7101.0223
89.0238; 59.0111		Perseitol
30.37191.0552191.0556−2.191.72C7H12O6-Quinic acid isomer a
40.44191.0551191.0556−2.698.94C7H12O6-Quinic acid isomer b
51.48315.1071315.108−2.999.93C14H20O8153.0197; 135.0442Hydroxytyrosol glucoside
62.57353.0858353.0873−4.299.99C16H18O9179.0302; 191.0532Caffeoylquinic acid isomer a
73.49337.0914337.0923−2.799.67C16H18O8163.0368; 191.0536Coumaroylquinic acid isomer a
83.89337.0909337.0923−4.299.99C16H18O8163.0363; 191.0515Coumaroylquinic acid isomer b
94.12443.1906443.1917−2.599.76C21H32O10113.0245; 101.0224Penstemide
104.58353.0868353.0873−1.499.99C16H18O9179.0334; 191.0531Caffeoylquinic acid isomer b
114.90337.0914337.0923−2.791.94C16H17O8163.0382; 191.0539Coumaroylquinic acid isomer c
125.02387.1641387.1655−3.699.95C18H28O9279.0305; 207.0988; 163.1096Tuberonic acid glucoside
135.22289.0698289.0712−4.899.63C15H14O6137.0219Catechin
145.34449.1082449.1084−0.4100C21H22O11287.0541Eriodictyol 7-O-glucoside
155.57337.0915337.0923−2.499.85C16H18O8163.0378; 191.0517Coumaroylquinic acid isomer d
165.62577.1329577.125213.390.86C19H30O20289.0682Procyanidin dimer
175.77415.1589415.1604−3.699.83C19H28O10339 1212-Phenylethyl beta-primeveroside
185.93463.2179463.21790.0100C21H36O11285.0396; 125.0228Luteolin/kaempferol derivative isomer a
196.85463.2164463.2179−3.299.71C21H36O11285.0390; 125.0202Luteolin/kaempferol derivative isomer b
206.981151.25071151.2575−5.994.31C46H56O34863.1739; 289.0672Procyanidin tretamer
217.10441.1741441.1761−4.599.97C21H30O10330.1303Hydroxyabscisic acid beta-D-glucoside
227.31863.183863.18230.890.3C45H36O18711.1354; 573.1021; 411.068; 289.0684Procyanidin trimer
237.79625.1409625.14050.697.84C27H30O17300.0247Quercetin-diglucoside isomer a
247.92625.1389625.1405−2.696.64C27H30O17300.0242Quercetin-diglucoside isomer b
258.30463.2182463.21790.6100C21H36O11285.0363; 125.0241Luteolin/kaempferol derivative isomer c
269.13439.1009439.1029−4.698.94C23H20O9287.0497Triacetoxy-methoxy-methylflavone isomer a
279.25439.1011439.1029−4.198.07C23H20O9287.0468Triacetoxy-methoxy-methylflavone isomer b
2810.07439.1037439.10291.8100C23H20O9287.0533Triacetoxy-methoxy-methylflavone isomer c
2910.66461.2382461.2387−1.1100C22H38O10285.0348Luteolin/kaempferol derivative II isomer a
3010.70461.2405461.23873.9100C22H38O10285.0439Luteolin/kaempferol derivative II isomer b
3111.19461.2397461.23872.2100C22H38O10285.0234Luteolin/kaempferol derivative II isomer c
3211.32461.2387461.23870.0100C22H38O10285.0355Luteolin/kaempferol derivative II isomer d
Table
Table 2. Log CFU/mL and pH of lactic acid bacteria in avocado seed expressed as average ± standard deviation.
Table 2. Log CFU/mL and pH of lactic acid bacteria in avocado seed expressed as average ± standard deviation.
LAB0 h24 h48 h
Log10 CFU/mLLog10 CFU/mLpHLog10 CFU/mLpH
P. acidilactici CECT 5765 T7.96 ± 0.027.14 ± 0.024.94.17 ± 0.034.9
P. pentosaceus CECT 4695 T7.57 ± 0.017.37 ± 0.064.94.10 ± 0.094.8
Leu. mesenteroides CECT 219 T7.14 ± 0.017.04 ± 0.045.24.03 ± 0.055.2
Le. brevis CECT 53547.05 ± 0.037.01 ± 0.065.04.04 ± 0.044.9
La. plantarum CECT 748 T7.95 ± 0.026.72 ± 0.024.93.88 ± 0.094.9
La. plantarum CECT 95677.93 ± 0.067.31 ± 0.024.66.71 ± 0.034.5
Table
Table 3. Phenolic and other polar compounds quantified by HPLC-ESI-TOF-MS in the fermented avocado seeds and a control. Results are expressed as average ± standard deviation.
Table 3. Phenolic and other polar compounds quantified by HPLC-ESI-TOF-MS in the fermented avocado seeds and a control. Results are expressed as average ± standard deviation.
µg/g d.w.
CompoundP. acidilactici CECT 5765TP. pentosaceus
CECT 923		Leu. mesenteroides CECT 219TLe. brevis
CECT 5354		La. plantarum
CECT 748T		La. plantarum
CECT 9567		Control
5-O-Galloyl-1,4-galactarolactone92.23 ± 1.26 a–c86.57 ± 1.55 b–d93.26 ± 1.46 a, b83.46 ± 5.96 d85.35 ± 0.93 c–d88.19 ± 0.38 a–d94.43 ± 1.02 a
Quinic acid isomer a107.68 ± 3.90 b, c108.03 ± 3.99 b, c98.56 ± 1.79 b, c112.11 ± 12.08 a, b102.87 ± 0.17 b, c123.77 ± 3.04 a96.89 ± 0.99 c
Quinic acid isomer b146.31 ± 1.93 a–c145.11 ± 1.31 a–c139.39 ± 3.36 c, d151.87 ± 3.85 a137.38 ± 0.52 d148.12 ± 4.09 a, b140.71 ± 1.35 b–d
Perseitol12.42 ± 0.02 b12.18 ± 0.14 b10.36 ± 0.20 c12.00 ± 0.35 b10.90 ± 0.22 c12.20 ± 0.22 b15.46 ± 0.05 a
Hydroxytyrosol glucoside2989.76 ± 3.64 a1547.58 ± 11.64 c1740.31 ± 13.67 b1574.06 ± 10.30 c886.69 ± 8.32 e128.44 ± 9.63 f1255.66 ± 18.34 d
Caffeoylquinic acid isomer a888.91 ± 1.04 e787.01 ± 6.32 f922.80 ± 5.00 d802.99 ± 2.25 f1109.82 ± 4.88 c1168.20 ± 12.57 a1133.69 ± 4.70 b
Caffeoylquinic acid isomer b507.57 ± 2.48 e449.37 ± 3.71 f520.10 ± 0.57 d421.71 ± 1.11 g569.72 ± 1.57 c652.06 ± 6.83 a605.25 ± 7.33 b
Coumaroylquinic acid isomer a302.09 ± 4.99 a318.50 ± 1.73 a273.33 ± 11.65 b238.75 ± 10.77 c195.23 ± 9.63 d194.92 ± 0.16 d210.88 ± 4.20 d
Coumaroylquinic acid isomer b374.82 ± 13.54 c341.59 ± 7.27 d399.01 ± 4.21 c301.14 ± 16.01 e468.46 ± 7.37 b514.91 ± 13.53 a490.79 ± 3.34 a, b
Coumaroylquinic acid isomer c148.94 ± 3.80 a147.85 ± 7.42 a141.21 ± 1.22 a119.56 ± 4.14 b109.00 ± 5.16 b111.02 ± 2.63 b116.19 ± 2.57 b
Coumaroylquinic acid isomer d170.50 ± 6.77 d153.54 ± 5.15 e188.37 ± 0.35 c141.33 ± 5.54 e207.35 ± 0.94 b227.54 ± 7.46 a213.18 ± 5.62 a, b
Penstemide50.45 ± 1.54 a52.79 ± 3.15 a55.24 ± 2.28 a46.60 ± 11.55 a56.91 ± 0.77 a58.34 ± 1.06 a58.77 ± 0.10 a
Catechin48.00 ± 0.17 a39.23 ± 2.03 b38.46 ± 2.47 b33.04 ± 4.68 b33.37 ± 0.79 b37.48 ± 1.78 b45.75 ± 1.22 a
Eriodictyol 7-O-glucoside7.85 ± 0.08 b, c7.99 ± 0.21 a–c8.47 ± 0.28 a–c7.33 ± 1.24 c9.31 ± 0.07 a8.81 ± 0.01 a, b8.63 ± 0.04 a–c
2-Phenylethyl beta-primeveroside10.86 ± 0.26 a11.24 ± 0.00 a11.25 ± 0.08 a10.68 ± 1.37 a11.22 ± 0.01 a12.00 ± 0.47 a11.55 ± 0.13 a
Procyanidin dimer20.94 ± 0.31 b18.58 ± 0.32 c, d21.83 ± 0.24 a, b18.00 ± 1.21 d19.96 ± 0.12 b, c23.04 ± 0.93 a23.39 ± 0.91 a
Procyanidin tetramer18.79 ± 1.34 c16.31 ± 0.24 c21.97 ± 0.99 b17.68 ± 0.19 c23.74 ± 0.15 a, b25.70 ± 1.53 a22.82 ± 1.33 b
Procyanidin trimer166.92 ± 13.88 b, c133.81 ± 14.92 c204.51 ± 19.56 a, b128.79 ± 8.68 c216.82 ± 1.29 a240.41 ± 17.46 a203.83 ± 11.81 a, b
Luteolin/kaempferol derivative isomer a6.55 ± 0.12 a6.64 ± 0.09 a6.77 ± 0.12 a6.30 ± 0.73 a6.52 ± 0.06 a6.90 ± 0.12 a6.93 ± 0.09 a
Luteolin/kaempferol derivative isomer b12.28 ± 0.73 a12.73 ± 0.34 a12.84 ± 0.33 a11.74 ± 1.56 a12.21 ± 0.26 a13.10 ± 0.42 a13.01 ± 0.02 a
Luteolin/kaempferol derivative isomer c5.53 ± 0.08 a5.72 ± 0.37 a5.79 ± 0.09 a5.55 ± 0.64 a5.63 ± 0.41 a5.82 ± 0.13 a6.05 ± 0.08 a
Quercetin-diglucoside isomer a4.95 ± 0.08 b, c5.00 ± 0.06 b, c5.38 ± 0.14 a–c4.72 ± 0.48 c6.05 ± 0.48 a5.52 ± 0.16 a, b5.42 ± 0.04 a–c
Quercetin-diglucoside isomer b5.00 ± 0.14 b, c5.13 ± 0.11 a–c5.41 ± 0.01 a–c4.87 ± 0.47 c5.54 ± 0.14 a, b5.58 ± 0.01 a5.51 ± 0.02 a, b
Triacetoxy-methoxy-methylflavone isomer a3.81 ± 0.07 c, d4.17 ± 0.02 b4.84 ± 0.24 a3.72 ± 0.19 d4.14 ± 0.00 b, c5.05 ± 0.04 a4.84 ± 0.03 a
Triacetoxy-methoxy-methylflavone isomer b3.88 ± 0.04 d3.91 ± 0.03 c, d4.24 ± 0.09 b3.84 ± 0.23 d4.19 ± 0.03 b, c4.54 ± 0.02 a4.39 ± 0.11 a, b
Triacetoxy-methoxy-methylflavone isomer c4.66 ± 0.13 d4.77 ± 0.14 c, d5.42 ± 0.29 a, b4.67 ± 0.36 c, d5.23 ± 0.13 b, c5.80 ± 0.06 a5.49 ± 0.12 a, b
Luteolin/kaempferol derivative II isomer a8.57 ± 0.43 b9.00 ± 0.67 a, b9.51 ± 0.18 a, b8.62 ± 1.20 b10.39 ± 0.07 a9.06 ± 0.13 a, b9.34 ± 0.20 a, b
Luteolin/kaempferol derivative II isomer b12.43 ± 0.67 b, c12.29 ± 0.14 b, c12.79 ± 0.05 b11.22 ± 1.02 c25.67 ± 0.10 a13.31 ± 0.03 b13.18 ± 0.15 b
Luteolin/kaempferol derivative II isomer c5.42 ± 0.03 b, c5.65 ± 0.02 b, c5.79 ± 0.02 a–c5.21 ± 0.58 c6.41 ± 0.04 a5.82 ± 0.14 a–c5.90 ± 0.01 a, b
Luteolin/kaempferol derivative II isomer d8.31 ± 0.54 a, b8.88 ± 0.55 a, b8.78 ± 0.02 a, b7.91 ± 0.89 b9.54 ± 0.20 a8.91 ± 0.55 a, b8.85 ± 0.00 a, b
Sum of phenolic acids and precursors5636.58 ± 42.08 a3998.58 ± 48.55 d4423.06 ± 41.82 b3863.52 ± 66.05 d, e3786.53 ± 38.55 e3268.97 ± 59.95 f4263.23 ± 48.44 c
Sum of procyanidins254.65 ± 15.70 b, c207.93 ± 17.52 c, d286.77 ± 23.26 a, b197.51 ± 14.75 d293.89 ± 2.34 a, b326.63 ± 21.70 a295.80 ± 15.27 a, b
Sum of others388.41 ± 9.96 a391.68 ± 14.97 a409.69 ± 8.12 a373.12 ± 47.29 a423.50 ± 8.16 a417.40 ± 6.17 a428.47 ± 6.19 a
Sum of polar compounds6279.63 ± 67.74 a4598.19 ± 81.04 c5119.52 ± 73.20 b4434.15 ± 128.09 c4503.92 ± 49.04 c4013.01 ± 87.81 d4987.50 ± 69.90 b
Different letters (a–g) in the same line indicate significant differences.
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Razola-Díaz, M.d.C.; De Montijo-Prieto, S.; Aznar-Ramos, M.J.; Jiménez-Valera, M.; Ruiz-Bravo, A.; Verardo, V.; Gómez-Caravaca, A.M. Effect of Lactic Acid Bacteria Fermentation on the Polar Compounds Content with Antioxidant and Antidiabetic Activity of Avocado Seed Extracts. Fermentation 2023, 9, 420. https://doi.org/10.3390/fermentation9050420

AMA Style

Razola-Díaz MdC, De Montijo-Prieto S, Aznar-Ramos MJ, Jiménez-Valera M, Ruiz-Bravo A, Verardo V, Gómez-Caravaca AM. Effect of Lactic Acid Bacteria Fermentation on the Polar Compounds Content with Antioxidant and Antidiabetic Activity of Avocado Seed Extracts. Fermentation. 2023; 9(5):420. https://doi.org/10.3390/fermentation9050420

Chicago/Turabian Style

Razola-Díaz, María del Carmen, Soumi De Montijo-Prieto, María José Aznar-Ramos, María Jiménez-Valera, Alfonso Ruiz-Bravo, Vito Verardo, and Ana María Gómez-Caravaca. 2023. "Effect of Lactic Acid Bacteria Fermentation on the Polar Compounds Content with Antioxidant and Antidiabetic Activity of Avocado Seed Extracts" Fermentation 9, no. 5: 420. https://doi.org/10.3390/fermentation9050420

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