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

Citation: 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 Academic Editors: Federica Tonolo and Maria Pia Rigobello Received: 21 March 2023 Revised: 18 April 2023 Accepted: 24 April 2023 Published: 27 April 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). fermentation Article Effect of Lactic Acid Bacteria Fermentation on the Polar Compounds Content with Antioxidant and Antidiabetic Activity of Avocado Seed Extracts María del Carmen Razola-Díaz 1,2,† , Soumi De Montijo-Prieto 3,† , María JoséAznar-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; [email protected] (M.d.C.R.-D.) 2 Biomedical Research Center, Institute of Nutrition and Food Technology ‘JoséMataix’, University of Granada, Avda del Conocimiento sn, 18100 Granada, Spain 3Department 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 *Correspondence: [email protected] † These authors contributed equally to this work. ‡ These authors are joint senior authors on this work. 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 µ g/g d.w.), and by extension, of hydroxytyrosol glucoside (2989.76 ± 3.64 µ g/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 µ g 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. Keywords: avocado seed waste; HPLC-ESI-TOF-MS; alpha-amylase; submerged fermentation; phenolic compounds 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] . Fermentation 2023,9, 420. https://doi.org/10.3390/fermentation9050420 https://www.mdpi.com/journal/fermentation Fermentation 2023,9, 420 2 of 17 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 – 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 Fermentation 2023,9, 420 3 of 17 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 MontijoPrieto 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; R 2 = 0.982), chlorogenic acid ( y = 101.17x −683.25 ; R 2 = 0.9933), ferulic acid (y = 10.502x + 353.77; R 2 = 0.9964), quercetin (y = 288.76x + 2462.1; R 2 = 0.9673), catechin (y = 315.87x − 415.59; R 2 = 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 µ L of fermented or non-fermented methanolic extract of avocado seed (20 mg/mL) was added to 100 µ L of porcine pancreatic alpha-amylase ( ≥ 2 µ d/mL) and pre-incubated during 5 min at 37 ◦ C. Then, 200 µ L 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)): %Inhibition =100 −Absorbance control −Absorbance sample Absorbance control ×100 (1) 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-TOFMS, 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 Fermentation 2023,9, 420 4 of 17 calculated m/z, time (min), error (ppm), score (%), molecular formula, m/zmain 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. Table 1. Polar compounds identified by HPLC-ESI-TOF-MS in fermented and non-fermented avocado seeds. No. Time (min) m/z Experimental m/z Calculated Error (ppm) Score (%) Molecular Formula m/zin Source Fragments Compound 1 0.26 343.0304 343.0301 0.9 94.75 C13H12O11 201.0226 5-O-Galloyl-1,4-galactarolactone 2 0.33 211.0813 211.0823 −4.7 91.29 C7H16O7101.0223 89.0238; 59.0111 Perseitol 3 0.37 191.0552 191.0556 −2.1 91.72 C7H12O6Quinic acid isomer a 4 0.44 191.0551 191.0556 −2.6 98.94 C7H12O6Quinic acid isomer b 5 1.48 315.1071 315.108 −2.9 99.93 C14H20O8153.0197; 135.0442 Hydroxytyrosol glucoside 6 2.57 353.0858 353.0873 −4.2 99.99 C16H18O9179.0302; 191.0532 Caffeoylquinic acid isomer a 7 3.49 337.0914 337.0923 −2.7 99.67 C16H18O8163.0368; 191.0536 Coumaroylquinic acid isomer a 8 3.89 337.0909 337.0923 −4.2 99.99 C16H18O8163.0363; 191.0515 Coumaroylquinic acid isomer b 9 4.12 443.1906 443.1917 −2.5 99.76 C21H32O10 113.0245; 101.0224 Penstemide 10 4.58 353.0868 353.0873 −1.4 99.99 C16H18O9179.0334; 191.0531 Caffeoylquinic acid isomer b 11 4.90 337.0914 337.0923 −2.7 91.94 C16H17O8163.0382; 191.0539 Coumaroylquinic acid isomer c 12 5.02 387.1641 387.1655 −3.6 99.95 C18H28O9279.0305; 207.0988; 163.1096 Tuberonic acid glucoside 13 5.22 289.0698 289.0712 −4.8 99.63 C15H14O6137.0219 Catechin 14 5.34 449.1082 449.1084 −0.4 100 C21H22O11 287.0541 Eriodictyol 7-O-glucoside 15 5.57 337.0915 337.0923 −2.4 99.85 C16H18O8163.0378; 191.0517 Coumaroylquinic acid isomer d 16 5.62 577.1329 577.1252 13.3 90.86 C19H30O20 289.0682 Procyanidin dimer 17 5.77 415.1589 415.1604 −3.6 99.83 C19H28O10 339 121 2-Phenylethyl beta-primeveroside 18 5.93 463.2179 463.2179 0.0 100 C21H36O11 285.0396; 125.0228 Luteolin/kaempferol derivative isomer a 19 6.85 463.2164 463.2179 −3.2 99.71 C21H36O11 285.0390; 125.0202 Luteolin/kaempferol derivative isomer b 20 6.98 1151.2507 1151.2575 −5.9 94.31 C46H56O34 863.1739; 289.0672 Procyanidin tretamer 21 7.10 441.1741 441.1761 −4.5 99.97 C21H30O10 330.1303 Hydroxyabscisic acid beta-D-glucoside 22 7.31 863.183 863.1823 0.8 90.3 C45H36O18 711.1354; 573.1021; 411.068; 289.0684 Procyanidin trimer 23 7.79 625.1409 625.1405 0.6 97.84 C27H30O17 300.0247 Quercetin-diglucoside isomer a 24 7.92 625.1389 625.1405 −2.6 96.64 C27H30O17 300.0242 Quercetin-diglucoside isomer b 25 8.30 463.2182 463.2179 0.6 100 C21H36O11 285.0363; 125.0241 Luteolin/kaempferol derivative isomer c 26 9.13 439.1009 439.1029 −4.6 98.94 C23H20O9287.0497 Triacetoxy-methoxy-methylflavone isomer a 27 9.25 439.1011 439.1029 −4.1 98.07 C23H20O9287.0468 Triacetoxy-methoxy-methylflavone isomer b 28 10.07 439.1037 439.1029 1.8 100 C23H20O9287.0533 Triacetoxy-methoxy-methylflavone isomer c 29 10.66 461.2382 461.2387 −1.1 100 C22H38O10 285.0348 Luteolin/kaempferol derivative II isomer a Fermentation 2023,9, 420 5 of 17 Table 1. Cont. No. Time (min) m/z Experimental m/z Calculated Error (ppm) Score (%) Molecular Formula m/zin Source Fragments Compound 30 10.70 461.2405 461.2387 3.9 100 C22H38O10 285.0439 Luteolin/kaempferol derivative II isomer b 31 11.19 461.2397 461.2387 2.2 100 C22H38O10 285.0234 Luteolin/kaempferol derivative II isomer c 32 11.32 461.2387 461.2387 0.0 100 C22H38O10 285.0355 Luteolin/kaempferol derivative II isomer d Fermentation 2023, 9, x FOR PEER REVIEW 5 of 17 31 11.19 461.2397 461.2387 2.2 100 C 22 H 38 O 10 285.0234 Luteolin/kaempferol derivative II isomer c 32 11.32 461.2387 461.2387 0.0 100 C 22 H 38 O 10 285.0355 Luteolin/kaempferol derivative II isomer d 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. Table 2. Log CFU/mL and pH of lactic acid bacteria in avocado seed expressed as average ± standard deviation. LAB 0 h 24 h 48 h Log 10 CFU/mL Log 10 CFU/mL pH Log 10 CFU/mL pH P. acidilactici CECT 5765 T 7.96 ± 0.02 7.14 ± 0.02 4.9 4.17 ± 0.03 4.9 P. pentosaceus CECT 4695 T 7.57 ± 0.01 7.37 ± 0.06 4.9 4.10 ± 0.09 4.8 Leu. mesenteroides CECT 219 T 7.14 ± 0.01 7.04 ± 0.04 5.2 4.03 ± 0.05 5.2 Le. brevis CECT 5354 7.05 ± 0.03 7.01 ± 0.06 5.0 4.04 ± 0.04 4.9 La. plantarum CECT 748 T 7.95 ± 0.02 6.72 ± 0.02 4.9 3.88 ± 0.09 4.9 La. plantarum CECT 9567 7.93 ± 0.06 7.31 ± 0.02 4.6 6.71 ± 0.03 4.5 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 (C 9 H 7 O 4 ) − of caffeic acid and 191 (C 7 H 11 O 6 ) − 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 (C 9 H 7 O 3 ) − and 191 (C 7 H 11 O 6 ) − , 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 (C 15 H 8 O 7 ) 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 (C 2 H 3 O 2 ) − , 89 (C 3 H 5 O 3 ) − , and 101 (C 4 H 5 O 3 ) − [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]. 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. Table 2. Log CFU/mL and pH of lactic acid bacteria in avocado seed expressed as average ± standard deviation. LAB 0 h 24 h 48 h Log10 CFU/mL Log10 CFU/mL pH Log10 CFU/mL pH P. acidilactici CECT 5765 T 7.96 ±0.02 7.14 ±0.02 4.9 4.17 ±0.03 4.9 P. pentosaceus CECT 4695 T 7.57 ±0.01 7.37 ±0.06 4.9 4.10 ±0.09 4.8 Leu. mesenteroides CECT 219 T 7.14 ±0.01 7.04 ±0.04 5.2 4.03 ±0.05 5.2 Le. brevis CECT 5354 7.05 ±0.03 7.01 ±0.06 5.0 4.04 ±0.04 4.9 La. plantarum CECT 748 T 7.95 ±0.02 6.72 ±0.02 4.9 3.88 ±0.09 4.9 La. plantarum CECT 9567 7.93 ±0.06 7.31 ±0.02 4.6 6.71 ±0.03 4.5 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 (C 9 H 7 O 4 ) − of caffeic acid and 191 (C 7 H 11 O 6 ) − 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/zin source fragments 163 (C 9 H 7 O 3 ) − and 191 (C 7 H 11 O 6 ) − , belonging to coumaric and quinic acids, respectively [18]. With the m/z625, two quercetin-diglucoside isomers named a and b were detected according to its m/z in source fragment 300 (C 15 H 8 O 7 ) 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]. Fermentation 2023,9, 420 6 of 17 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/zin source fragments 59 (C2H3O2)−, 89 (C3H5O3)−, and 101 (C4H5O3)−[18]. The iridoid penstemide was identified corresponding to peak 9 with the m/zof 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 C 14 H 20 O 8 , the m/z315, and the m/z in source fragment 153 that corresponds to hydroxytyrosol [18]. At 7.20 min, the compound found with the m/z441 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/z343, 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/z449 and the predicted formula C 21 H 22 O 11 . It was named as eriodictyol 7-O-glucoside, in concordance with its main m/zin source fragment 287 (C 15 H 11 O 6 ) − , 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/z439, three flavonoid isomers were identified and called triacetoxymethoxy-methylflavone isomer a, b, and c, according to the PUBCHEM database (PubChem CID: 129815511). Its main m/z in source fragment 287 (C 15 H 11 O 6 ) − 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/z463 (peaks 18, 19, and 25) and the m/z in source fragments 285 (C 15 H 9 O 6 ) − and 125 (C 6 H 9 O 5 ) − , 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/zin source fragment 285 (C 15 H 9 O 6 ) − 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 Fermentation 2023,9, 420 7 of 17 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 Log 10 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 Log 10 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 Log 10 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 Log 10 CFU/mL (r = 0.9278 and 0.8662, respectively). Furthermore, perseitol, catechin, and hydrotytyrosol glucoside showed a significant positive correlation with the Log 10 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 Log 10 CFU/mL (r from − 0.7225 to − 0.9995). For procyanidin dimer, trimer, and tetramer, the opposite correlation was found with Log 10 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 Log 10 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 Log 10 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. Fermentation 2023,9, 420 8 of 17 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. Compound P. acidilactici CECT 5765T P. pentosaceus CECT 923 Leu. mesenteroides CECT 219T Le. brevis CECT 5354 La. plantarum CECT 748T La. plantarum CECT 9567 Control 5-O-Galloyl-1,4-galactarolactone 92.23 ±1.26 a–c 86.57 ±1.55 b–d 93.26 ±1.46 a, b 83.46 ±5.96 d 85.35 ±0.93 c–d 88.19 ±0.38 a–d 94.43 ±1.02 a Quinic acid isomer a 107.68 ±3.90 b, c 108.03 ±3.99 b, c 98.56 ±1.79 b, c 112.11 ±12.08 a, b 102.87 ±0.17 b, c 123.77 ±3.04 a 96.89 ±0.99 c Quinic acid isomer b 146.31 ±1.93 a–c 145.11 ±1.31 a–c 139.39 ±3.36 c, d 151.87 ±3.85 a 137.38 ±0.52 d 148.12 ±4.09 a, b 140.71 ±1.35 b–d Perseitol 12.42 ±0.02 b 12.18 ±0.14 b 10.36 ±0.20 c 12.00 ±0.35 b 10.90 ±0.22 c 12.20 ±0.22 b 15.46 ±0.05 a Hydroxytyrosol glucoside 2989.76 ±3.64 a 1547.58 ±11.64 c 1740.31 ±13.67 b 1574.06 ±10.30 c 886.69 ±8.32 e 128.44 ±9.63 f 1255.66 ±18.34 d Caffeoylquinic acid isomer a 888.91 ±1.04 e 787.01 ±6.32 f 922.80 ±5.00 d 802.99 ±2.25 f 1109.82 ±4.88 c 1168.20 ±12.57 a 1133.69 ±4.70 b Caffeoylquinic acid isomer b 507.57 ±2.48 e 449.37 ±3.71 f 520.10 ±0.57 d 421.71 ±1.11 g 569.72 ±1.57 c 652.06 ±6.83 a 605.25 ±7.33 b Coumaroylquinic acid isomer a 302.09 ±4.99 a 318.50 ±1.73 a 273.33 ±11.65 b 238.75 ±10.77 c 195.23 ±9.63 d 194.92 ±0.16 d 210.88 ±4.20 d Coumaroylquinic acid isomer b 374.82 ±13.54 c 341.59 ±7.27 d 399.01 ±4.21 c 301.14 ±16.01 e 468.46 ±7.37 b 514.91 ±13.53 a 490.79 ±3.34 a, b Coumaroylquinic acid isomer c 148.94 ±3.80 a 147.85 ±7.42 a 141.21 ±1.22 a 119.56 ±4.14 b 109.00 ±5.16 b 111.02 ±2.63 b 116.19 ±2.57 b Coumaroylquinic acid isomer d 170.50 ±6.77 d 153.54 ±5.15 e 188.37 ±0.35 c 141.33 ±5.54 e 207.35 ±0.94 b 227.54 ±7.46 a 213.18 ±5.62 a, b Penstemide 50.45 ±1.54 a 52.79 ±3.15 a 55.24 ±2.28 a 46.60 ±11.55 a 56.91 ±0.77 a 58.34 ±1.06 a 58.77 ±0.10 a Catechin 48.00 ±0.17 a 39.23 ±2.03 b 38.46 ±2.47 b 33.04 ±4.68 b 33.37 ±0.79 b 37.48 ±1.78 b 45.75 ±1.22 a Eriodictyol 7-O-glucoside 7.85 ±0.08 b, c 7.99 ±0.21 a–c 8.47 ±0.28 a–c 7.33 ±1.24 c 9.31 ±0.07 a 8.81 ±0.01 a, b 8.63 ±0.04 a–c 2-Phenylethyl beta-primeveroside 10.86 ±0.26 a 11.24 ±0.00 a 11.25 ±0.08 a 10.68 ±1.37 a 11.22 ±0.01 a 12.00 ±0.47 a 11.55 ±0.13 a Procyanidin dimer 20.94 ±0.31 b 18.58 ±0.32 c, d 21.83 ±0.24 a, b 18.00 ±1.21 d 19.96 ±0.12 b, c 23.04 ±0.93 a 23.39 ±0.91 a Procyanidin tetramer 18.79 ±1.34 c 16.31 ±0.24 c 21.97 ±0.99 b 17.68 ±0.19 c 23.74 ±0.15 a, b 25.70 ±1.53 a 22.82 ±1.33 b Procyanidin trimer 166.92 ±13.88 b, c 133.81 ±14.92 c 204.51 ±19.56 a, b 128.79 ±8.68 c 216.82 ±1.29 a 240.41 ±17.46 a 203.83 ±11.81 a, b Luteolin/kaempferol derivative isomer a 6.55 ±0.12 a 6.64 ±0.09 a 6.77 ±0.12 a 6.30 ±0.73 a 6.52 ±0.06 a 6.90 ±0.12 a 6.93 ±0.09 a Luteolin/kaempferol derivative isomer b 12.28 ±0.73 a 12.73 ±0.34 a 12.84 ±0.33 a 11.74 ±1.56 a 12.21 ±0.26 a 13.10 ±0.42 a 13.01 ±0.02 a Fermentation 2023,9, 420 9 of 17 Table 3. Cont. µg/g d.w. Compound P. acidilactici CECT 5765T P. pentosaceus CECT 923 Leu. mesenteroides CECT 219T Le. brevis CECT 5354 La. plantarum CECT 748T La. plantarum CECT 9567 Control Luteolin/kaempferol derivative isomer c 5.53 ±0.08 a 5.72 ±0.37 a 5.79 ±0.09 a 5.55 ±0.64 a 5.63 ±0.41 a 5.82 ±0.13 a 6.05 ±0.08 a Quercetin-diglucoside isomer a 4.95 ±0.08 b, c 5.00 ±0.06 b, c 5.38 ±0.14 a–c 4.72 ±0.48 c 6.05 ±0.48 a 5.52 ±0.16 a, b 5.42 ±0.04 a–c Quercetin-diglucoside isomer b 5.00 ±0.14 b, c 5.13 ±0.11 a–c 5.41 ±0.01 a–c 4.87 ±0.47 c 5.54 ±0.14 a, b 5.58 ±0.01 a 5.51 ±0.02 a, b Triacetoxy-methoxy-methylflavone isomer a 3.81 ±0.07 c, d 4.17 ±0.02 b 4.84 ±0.24 a 3.72 ±0.19 d 4.14 ±0.00 b, c 5.05 ±0.04 a 4.84 ±0.03 a Triacetoxy-methoxy-methylflavone isomer b 3.88 ±0.04 d 3.91 ±0.03 c, d 4.24 ±0.09 b 3.84 ±0.23 d 4.19 ±0.03 b, c 4.54 ±0.02 a 4.39 ±0.11 a, b Triacetoxy-methoxy-methylflavone isomer c 4.66 ±0.13 d 4.77 ±0.14 c, d 5.42 ±0.29 a, b 4.67 ±0.36 c, d 5.23 ±0.13 b, c 5.80 ±0.06 a 5.49 ±0.12 a, b Luteolin/kaempferol derivative II isomer a 8.57 ±0.43 b 9.00 ±0.67 a, b 9.51 ±0.18 a, b 8.62 ±1.20 b 10.39 ±0.07 a 9.06 ±0.13 a, b 9.34 ±0.20 a, b Luteolin/kaempferol derivative II isomer b 12.43 ±0.67 b, c 12.29 ±0.14 b, c 12.79 ±0.05 b 11.22 ±1.02 c 25.67 ±0.10 a 13.31 ±0.03 b 13.18 ±0.15 b Luteolin/kaempferol derivative II isomer c 5.42 ±0.03 b, c 5.65 ±0.02 b, c 5.79 ±0.02 a–c 5.21 ±0.58 c 6.41 ±0.04 a 5.82 ±0.14 a–c 5.90 ±0.01 a, b Luteolin/kaempferol derivative II isomer d 8.31 ±0.54 a, b 8.88 ±0.55 a, b 8.78 ±0.02 a, b 7.91 ±0.89 b 9.54 ±0.20 a 8.91 ±0.55 a, b 8.85 ±0.00 a, b Sum of phenolic acids and precursors 5636.58 ±42.08 a 3998.58 ±48.55 d 4423.06 ±41.82 b 3863.52 ± 66.05 d, e 3786.53 ±38.55 e 3268.97 ±59.95 f 4263.23 ±48.44 c Sum of procyanidins 254.65 ±15.70 b, c 207.93 ±17.52 c, d 286.77 ±23.26 a, b 197.51 ±14.75 d 293.89 ±2.34 a, b 326.63 ±21.70 a 295.80 ±15.27 a, b Sum of others 388.41 ±9.96 a 391.68 ±14.97 a 409.69 ±8.12 a 373.12 ±47.29 a 423.50 ±8.16 a 417.40 ±6.17 a 428.47 ±6.19 a Sum of polar compounds 6279.63 ±67.74 a 4598.19 ±81.04 c 5119.52 ±73.20 b 4434.15 ±128.09 c 4503.92 ±49.04 c 4013.01 ±87.81 d 4987.50 ±69.90 b Different letters (a–g) in the same line indicate significant differences. 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