Solid-State Fermented Pineapple Peel: A Novel Food Ingredient with Antioxidant and Anti-Inflammatory Properties

It has been reported that pineapple (Ananas comosus) contains healthy nutrients and phytochemicals associated with antioxidant and anti-inflammatory capacities. However, a substantial amount of pineapple residue is produced due to a lack of valorization applications at the industrial scale, resulting in the loss of valuable nutrients. Solid-state fermentation (SSF) is proposed as an innovative strategy to enhance the release of bound phenolics from pineapple residues. In this work, the effects of SSF of pineapple peels with Lactobacillus plantarum, Lactobacillus rhamnosus, and Aspergillus oryzae on the release of phenolic compounds and their antioxidant and anti-inflammatory activities were evaluated, respectively. Pineapple peel extracts after SSF showed an increase in the release of phenolic compounds (248.11% with L. plantarum, 182% with A. oryzae, and 180.10% with L. rhamnosus), which led to an increase in the cellular antioxidant (81.94% with L. rhamnosus) and anti-inflammatory potential (nitric oxide inhibition of 62% with L. rhamnosus) compared to non-fermented extracts. Therefore, SSF of pineapple peels with L. plantarum, L. rhamnosus, and A. oryzae thrives as a new approach for the production of secondary metabolites with remarkable biological benefits, which can be the precursors for novel biofortified and nutraceutical-enriched foods that meet the needs of the most demanding and health-conscious consumers.


Introduction
Waste disposal is a significant issue for many agro-industries since it is highly susceptible to microbial decomposition and causes substantial environmental problems.According to the Food and Agriculture Organization of the United Nations (FAO), just around 14% of the food produced was lost from the post-harvest stage in the world [1].Using agroindustrial waste by conversion into value-added products may be an excellent solution to environmental pollution [2].
Pineapple (Ananas comosus), the only edible member of the family Bromeliaceae, is widely cultivated in several tropical countries, including the Philippines, Thailand, Indonesia, Malaysia, Kenya, India, China, and South America [3].Pineapple is usually consumed as fresh pulp or processed into different products, including jams, purees, or canned juices [4].However, about 80% of the total fruit weight in the form of the crown, outer peel, and core is discarded, causing a waste disposal problem of about 22.5 million tons of pineapple annually [5,6].These residues are rich in cellulose, hemicellulose, and phenolic compounds, which have been recognized as antioxidants and for preventing chronic inflammation, cardiovascular disease (CVD), cancer, and diabetes [7].In addition, pineapple residues are a source of polyphenols with strong antioxidant activities.Most phenolic compounds occur primarily in conjugated form, bound to the matrix, making it difficult to extract or liberate them [7].Since the resonance stabilization of free radicals depends on the presence of free hydroxyl groups on the phenolic rings, these conjugations diminish their capacity to function as effective antioxidants [8].
The pretreatment of agro-industrial residues with solid-state fermentation (SSF) system technology could improve the recovery of phenolic compounds through the hydrolysis of these conjugates with microorganism-produced degrading enzymes [9].SSF consists of using moist substrates for a microbial culture in the near absence of available water [10].For example, Lactobacillus plantarum and Lactobacillus rhamnosus are microorganisms that have been used in many SSF studies because of their ability to synthesize hydrolytic enzymes [11][12][13][14].
Aspergillus oryzae is an important food-grade filamentous fungus that has been used in fermentation technologies for the preparation of traditional Asian fermented foods, such as sake, miso, and shoyu [15].In SSF processes, high amounts of β-glucosidase can be produced, which plays a crucial role in the hydrolysis of phenolic glycosides [16].
Since studies on using pineapple waste as a substrate in SSF systems for producing added-value compounds are limited, further research is necessary.
Therefore, this study aims to evaluate the feasibility of pineapple residue as a substrate for producing phenolic compounds with antioxidant and anti-inflammatory activities by L. plantarum, L. rhamnosus, and A. oryzae via SSF.broth at 37 • C for 24 h.Bacterial strains were subcultured two more times, repeating the last step.Then, strains were centrifuged (4000× g, 5 min, 4 • C), washed using a sterile 0.85% w/v NaCl solution, and adjusted to a final concentration of 7 log colony-forming unit CFU/mL.Cell density was measured by optical density (OD) using a spectrophotometer (Genesys 10S, Thermo Scientific, Waltham, MA, USA) at 600 nm.

Preparation of Fungal Inoculum
A. oryzae spores (ATCC 22788) obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were inoculated following the method reported by Villasante et al. [18].Spores were cultured in potato dextrose agar and incubated at 30 • C for 5 days.Spores were collected using 10 mL of 0.1% v/v Tween 80 solution with distilled water and counted using a hemocytometer (Bright-Line, Hausser Scientific, Horshara, PA, USA) in a microscope (Olympus CK2, Marshall Scientific, Hampton, NH, USA).Spores were diluted to reach a concentration of 5 log spores/mL.

Solid-State Fermentation with L. plantarum and L. rhamnosus
For the solid-state fermentation of pineapple peels using lactic acid bacteria, 5 mL of L. plantarum and L. rhamnosus 0.85% sodium chloride (NaCl) solution was inoculated into 5 g of pineapple peels previously sterilized with ultraviolet light for 30 min to ensure the absence of other bacteria and fungi.Water activity (Aw) of L. plantarum and L. rhamnosus was determined using an AquaLab Dew Point Water Activity Meter 4TE (Decagon Devices, Pullman, WA, USA) at 24 • C. Moisture content during fermentation was 88%.
The fermentation process was performed in triplicate for 5 days at 37 • C in an incubator (Shel Lab 1535, Sheldon Manufacturing Inc., Cornelius, OR, USA).The initial pH of fermented pineapple was adjusted to 4. Samples were collected at 24 h intervals and stored at −80 • C for analysis of phenolic compounds and bioactivity.

Solid-State Fermentation with A. oryzae
For the solid-state fermentation of pineapple peels using A. oryzae, 5 mL of 0.1% Tween 20 solution was inoculated into 5 g of freeze-dried pineapple peels previously sterilized with ultraviolet light for 30 min [18].Water activity (Aw) of A. oryzae was determined using an AquaLab Dew Point Water Activity Meter 4TE (Decagon Devices, Pullman, WA, USA) at 24 • C. Moisture content during fermentation was 88%.
The fermentation process was performed in triplicate for 5 days at 30 • C in an incubator (Shel Lab 1535, Sheldon Manufacturing Inc., Cornelius, OR, USA).The initial pH of fermented pineapple was adjusted to 4. Samples were collected at 24 h intervals and stored at −80 • C for analysis of phenolic compounds and bioactivity.

Phenolic Compounds Extraction
After the solid-state fermentation of pineapple peels, phenolic compound extraction was performed using a slightly modified protocol from Acosta-Estrada et al. [19].Briefly, a methanol (80%) solution was added to previously freeze-dried solid-state fermentation samples in a proportion of 1:20 (w/v).Then, samples were vortexed for 1 min and stirred at 250 rpm for 10 min at 25 • C using an orbital shaker (Incubator 3500I, VWR International, Radnor, PA, USA).Samples were centrifuged (3000× g, 10 min, 4 • C), and the supernatant was recovered, freeze-dried (LABCONCO, Kansas City, MO, USA), and stored at −80 • C for further analysis.

HPLC-DAD Analysis of Phenolic Compounds
Fermented pineapple peel extracts (FPPE) were solubilized in 80% methanol and analyzed through high-performance liquid chromatography with diode-array detection (HPLC-DAD) according to the method reported by Steingass et al. [20].Analyses of phenolic compounds were performed using a high-performance liquid chromatography system coupled with a diode-array detector (HPLC-DAD) (1260 Infinity, Agilent Technologies, Santa Clara, CA, USA).The column used to separate phenolic compounds was a Luna C18(2) Phenomenex™ (250 × 4.6 mm, 5 µm particle size).Water (A) and methanol containing 1% (v/v) formic acid (B) were used as the mobile phase.The following gradients were used: 5 to 40% B (35 min), 40 to 70% B (15 min), 70 to 100% B (2 min), and isocratic hold at 100% B (3 min).The column was flushed back to 5% B (2 min) and held isocratically for 8 min.The total run time was 65 min at a flow rate of 0.8 mL/min and an oven temperature of 30 • C. The injection volume was 10 µL.The detection wavelengths were set to 280, 320, and 360 nm.Chromatographic data were processed with OpenLAB CDS ChemStation software version 1.8 (Agilent Technologies, Santa Clara, CA, USA).
Mass spectra of phenolic compounds in FPPE were identified according to the method reported by Steingass et al. [20], following the abovementioned method.It was performed using liquid chromatography time-of-flight mass spectrometry (LC/MS-TOF) coupled with electrospray ionization (ESI) (Agilent 1100 system, Agilent Technologies) using the same conditions for the HPLC-DAD analysis.Mass spectra were scanned in a range of m/z 100-1500 through positive electrospray ionization [ESI (+)].Nitrogen served as dry gas at a flow rate of 13 L/min and nebulizing gas at a pressure of 45 psi.The gas temperature was set to 350 • C, and the capillary potential was 4000 V.The fragmentation amplitude was set to 120 V. Mass Hunter Software version A.02.00 2005 (Agilent Technologies, Santa Clara, CA, USA).Analyst QS 1.1 Software (Applied Biosystems, Waltham, MA, USA) was used for the identification of compounds present in FPPE.
The identification of phenolic compounds was based on the retention time, DAD spectra, and their mass-to-charge (m/z) ratio.Quantification of phenolic compounds was performed using gallic acid, p-coumaric acid, ferulic acid, and quercetin as standards.Results were expressed as µg equivalents of each phenolic compound per g of FPPE in dry weight (DW).

Cellular Antioxidant Capacity Assay
The cellular antioxidant capacity assay was carried out according to the methodology of Ortega-Hernández et al. [23].Briefly, Caco2 cells were cultured in 96-well plates (5 × 10 4 cells/well) and allowed to adhere for 24 h.Then, cells were washed with PBS solution (pH 7.4) and treated with 100 µL of FPPE (25 µg/mL) containing DCFH-DA (60 µM).After incubation at 37 • C for 20 min, the treatment solutions were removed, and the cells were washed twice with a PBS solution.Finally, 100 µL of 500 µM AAPH solution was added to each well, except for the blank and negative control wells.Fluorescence emitted at 538 nm with excitation at 485 nm was measured with a microplate reader (Synergy HT, Bio-Tek, Winooski, VM, USA) every 2 min for 90 min at 37 • C.
The CAA values were calculated using Equation (3): where SA is the integrated area under the sample fluorescence versus time curve and CA is the integrated area of the control curve.

Cellular Anti-Inflammatory Potential Assay
The anti-inflammatory potential of the extracts was performed using the method proposed by Ortega-Hernández et al. [23].Raw 264.7 cells were cultured in 96-well plates (5 × 10 4 cells/well) and allowed to adhere for 24 h.Then, cells were treated with 50 µL of FPPE (25 µg/mL) and incubated for 4 h.Following, half of the wells were stimulated with lipopolysaccharide (LPS) at 1 µg/mL while the other half was used as the control for each sample.After 24 h of incubation, the nitrite concentration in the medium (100 µL) was measured at 550 nm (Synergy HT, Bio-Tek, Winooski, VM, USA).The nitric oxide (NO) production was measured using a nitrite standard curve (1.5-50 µM).

Measurement of Raw 264.7 Cell Viability
Cell viability was tested using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA).Absorbance values were read with a 96-well microplate reader (Synergy HT, Bio-Tek, Winooski, VM, USA) at 490 nm.The percentage (%) of cell viability was calculated by dividing the absorbance of treated cells by the absorbance of the control (untreated) cells.2.11.5.Measurement of COX-2, IL-1β, IL-2, IL-6, IL-10, and TNF-α The effect of FPPE on proinflammatory and anti-inflammatory cytokines was evaluated in Raw 264.7 cells.After performing the cellular anti-inflammatory potential assay, Raw 264.7 cells were lysed using 0.5% (v/v) Triton X-100 for 2 h.After incubation, lysates were mixed with 100 µL of PBS and centrifuged (2000× g, 5 min, 4 • C).Supernatants were recov-ered and stored at −80 • C until use.COX-2 and IL-10 were measured using a Human/Mouse Total COX-2 DuoSet IC and Mouse IL-10 ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.The absorbance values of cytokines were measured using a Synergy HT plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at 450 nm.Likewise, a MILLIPLEX MAP Mouse Cytokine/Chemokine panel was used to measure IL-1β, IL-2, IL-6, and TNF-α in the supernatant on a Luminex R 200 TM System with xPONENT@3.0software (Luminex, Austin, TX, USA).From the immunoassay, median fluorescent intensity (MFI) data using a polynomial curve-fitting method were used to calculate cytokine concentrations as per the manufacturers' guidelines.

Statistical Analysis and Data Processing
All results were expressed as the mean ± standard deviation, and all measurements were performed at least in triplicate.Statistical analyses were performed with the JMP Pro 16.0 software (SAS Institute Inc., Cary, NC, USA).One-way and two-way ANOVA were performed for the results obtained and were considered statistically significant at 95% confidence (p ≤ 0.05).Differences between treatments were analyzed by Tukey tests and were considered statistically significant at 95% confidence (p ≤ 0.05).

Pineapple Peel Bromatological Analysis and Chemical Composition
The chemical composition of pineapple peels is shown in Table S1.Pineapple peels show a great amount of available carbohydrates (42.29%) and dietary fiber (30.20%).These results are in agreement with previous reports, where the pineapple peel's chemical composition was 55.52% carbohydrates, 4.39% ashes, and 14.80% crude fiber [24].

Solid-State Fermentation of Pineapple Peels with L. plantarum, L. rhamnosus, and A. oryzae
During solid-state fermentation of pineapple peel, the results demonstrate that there were no pH or Aw changes during the fermentation process (Figure S1).It is important to measure these parameters since the growth of microorganisms and the production of enzymes depend on their variation [25][26][27].
The optimal pH ranges for the growth of L. plantarum and L. rhamnosus are between 4 and 9, respectively, while for A. oryzae, the optimal pH values fall within the range of 3.8 to 6 [28][29][30].Lower pH values could suggest variations in the production of phenolic compounds, as well as variances in the production of other bioactive compounds aside from phenolic compounds [27].Therefore, it is important to monitor these values to ensure that pH values during solid-state fermentation are not getting lower, thereby avoiding the production of non-valuable compounds.

Total Phenolic Content
Phenolics constitute the most prevalent secondary metabolites in plants, characterized by a structure containing an aromatic ring with at least one hydroxyl substituent [21].The total phenolic content of pineapple peels treated with solid-state fermentation was investigated.
Changes in total phenolic content (TPC) can be observed in Figure 1.The results show a significant increase in the TPC content in pineapple peels after five days of solidstate fermentation across all treatment groups.Notably, L. plantarum (LP) exhibits the highest release of TPC, with a significant difference compared to the other treatments (p ≤ 0.05), showing a significant increase of 248.11% on the fifth day of fermentation.This was followed by A. oryzae (AO) with an 182% increase and L. rhamnosus (LR) with a 158.44% increase on the fifth day of fermentation, all compared to the TPC content in pineapple peels on day 0. As previously reported, phenolic compounds are usually bound to cell wall structural components such as cellulose, hemicellulose, lignin, pectin, and rod-shaped structural proteins [7,31].The higher release of TPC with L. plantarum can be attributed to the enzymes produced during the fermentation processes (e.g., amylase, β-glucosidase, decarboxylase, lactate, dehydrogenase, peptidase, phenolic acid decarboxylase, phenol reductase, proteinase, TanA (tanALp), TanB (tanBLp) esterases).These enzymes can hydrolyze glucosides and break down plant cell walls, liberating phenolic compounds that were initially bound to these plant components [32][33][34].Conversely, the lower TPC production observed with L. rhamnosus and A. oryzae may be attributed to the distinct enzymes generated during their respective fermentation processes [32,34].
Regarding A. oryzae, previous studies have indicated that it secretes enzymes like αamylase, β-glucosidase, and cellulase during solid-state fermentation, contributing to the release of bound phenolic compounds [18,35,36].However, enzyme production by A. oryzae typically begins to rise between the fourth and fifth days of fermentation, as was reported in previous studies [37][38][39].This timing agrees with the observed increase in phenolic compound production in the current study.

HPLC-DAD and LC/MS-TOF Bioactive Compounds Quantification and Characterization
The profile of bioactive compounds obtained from the fermented pineapple peel extracts (FPPE) in L. rhamnosus, L. plantarum, and A. oryzae FPPE is shown in Table 1.Thirty compounds were identified in both the control and treated samples.The phenolic profile of pineapple peel agrees with previous reports [20,40].However, the concentration of identified individual phenolics varied with the effect of the applied treatment and fermentation time (Table 2).As previously reported, phenolic compounds are usually bound to cell wall structural components such as cellulose, hemicellulose, lignin, pectin, and rod-shaped structural proteins [7,31].The higher release of TPC with L. plantarum can be attributed to the enzymes produced during the fermentation processes (e.g., amylase, β-glucosidase, decarboxylase, lactate, dehydrogenase, peptidase, phenolic acid decarboxylase, phenol reductase, proteinase, TanA (tanALp), TanB (tanBLp) esterases).These enzymes can hydrolyze glucosides and break down plant cell walls, liberating phenolic compounds that were initially bound to these plant components [32][33][34].Conversely, the lower TPC production observed with L. rhamnosus and A. oryzae may be attributed to the distinct enzymes generated during their respective fermentation processes [32,34].
Regarding A. oryzae, previous studies have indicated that it secretes enzymes like α-amylase, β-glucosidase, and cellulase during solid-state fermentation, contributing to the release of bound phenolic compounds [18,35,36].However, enzyme production by A. oryzae typically begins to rise between the fourth and fifth days of fermentation, as was reported in previous studies [37][38][39].This timing agrees with the observed increase in phenolic compound production in the current study.

HPLC-DAD and LC/MS-TOF Bioactive Compounds Quantification and Characterization
The profile of bioactive compounds obtained from the fermented pineapple peel extracts (FPPE) in L. rhamnosus, L. plantarum, and A. oryzae FPPE is shown in Table 1.Thirty compounds were identified in both the control and treated samples.The phenolic profile of pineapple peel agrees with previous reports [20,40].However, the concentration of identified individual phenolics varied with the effect of the applied treatment and fermentation time (Table 2).Changes in individual bioactive compound content can be observed in Table 2.Ten of these compounds were only detected in the control sample.
These studies demonstrate the presence of phenolic acids after enzymatic β-glucosidase hydrolysis, suggesting that the hexosides initially present in pineapple peel are transformed into phenolic acids through the action of this enzyme.This observation supports the notion that the microorganisms employed in the solid-state fermentation of pineapple peel in this study produce the same enzyme, thereby explaining the variations in the profiles of phenolic compounds when compared to the FPPE profiles at day zero of fermentation.

2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Scavenging Activity
As it is known, many biologically active molecules in plants may contribute to antioxidant capacities, and pineapple is not an exception.The antioxidant activity of FPPE was determined in terms of the proportion (%) of DPPH scavenged, and values are shown in Figure 2.
In the case of L. rhamnosus (Figure 2A), a significant increase in scavenging activity was observed in L. rhamnosus FPPE in a time-dose-dependent manner when using concentrations of 250 and 500 µg/mL.Conversely, at a concentration of 1000 µg/mL of L. rhamnosus FPPE, scavenging activity exhibits a significant increase (50.05%) after just one day of fermentation compared to the control (p ≤ 0.05), and this enhanced activity was maintained without significant differences until day 5.
Furthermore, the highest induced antioxidant activity with L. rhamnosus treatment was observed after 4 d of fermentation.It was observed that a significant increase of 238.52%, 181.73%, and 62.44% was achieved with concentrations of 250 µg/mL, 500 µg/mL, and 1000 µg/mL FPPE, respectively, in comparison to their respective control groups.
Regarding L. plantarum, the scavenging activity (%) of FPPE also displays a timedependent increase (Figure 2B).At a concentration of 250 µg/mL of L. plantarum FPPE, scavenging activity exhibits a significant increment (48.52%) after one day of fermentation compared to the control (p ≤ 0.05).However, this heightened activity was consistently sustained on days 2, 3, and 4, with no significant variations.On the other hand, concentrations of 500 µg/mL and 1000 µg/mL FPPE show a significant daily increase in scavenging activity (%), reaching the highest level after 5 d of fermentation (155.74% and 61.65%, respectively).
Nevertheless, the fermentation treatments did not induce an immediate increment in the radical scavenging activity of pineapple peels (Figure 2C).

2,2-. Diphenyl-1-Picrylhydrazyl (DPPH) Scavenging Activity
As it is known, many biologically active molecules in plants may contribute to antioxidant capacities, and pineapple is not an exception.The antioxidant activity of FPPE was determined in terms of the proportion (%) of DPPH scavenged, and values are shown in Figure 2. In the case of L. rhamnosus (Figure 2A), a significant increase in scavenging activity was observed in L. rhamnosus FPPE in a time-dose-dependent manner when using concentrations of 250 and 500 µg/mL.Conversely, at a concentration of 1000 µg/mL of L. rhamnosus FPPE, scavenging activity exhibits a significant increase (50.05%) after just one day of fermentation compared to the control (p ≤ 0.05), and this enhanced activity was maintained without significant differences until day 5.
Furthermore, the highest induced antioxidant activity with L. rhamnosus treatment was observed after 4 d of fermentation.It was observed that a significant increase of 238.52%, 181.73%, and 62.44% was achieved with concentrations of 250 µg/mL, 500 µg/mL, and 1000 µg/mL FPPE, respectively, in comparison to their respective control groups.
Regarding L. plantarum, the scavenging activity (%) of FPPE also displays a time-dependent increase (Figure 2B).At a concentration of 250 µg/mL of L. plantarum FPPE, scavenging activity exhibits a significant increment (48.52%) after one day of fermentation compared to the control (p ≤ 0.05).However, this heightened activity was consistently When the interaction between microorganisms and fermentation times at 1000 µg/mL is compared (Figure 2D), results show that the radical scavenging activities of pineapple peel extracts fermented by L. plantarum and L. rhamnosus were not statistically different on days 3, 4, and 5 (p ≤ 0.05).Moreover, L. plantarum and L. rhamnosus FPPE at 1000 µg/mL concentrations had better DPPH scavenging activity compared to A. oryzae FPPE at day 5 at the same concentration (57.51% and 56.02%, respectively).
The results obtained from antioxidant activity assays of FPPE correlate with the increase in TPC of FPPE over time.These findings are consistent with prior studies evaluating the antioxidant activity of various pineapple residue extracts using the DPPH scavenging activity assay.For instance, Othman et al. [44] reported a 26.24% DPPH scavenging activity at a 500 µg/mL concentration of pineapple peel extract.In addition, de Oliveira et al. [45] reported an increase in DPPH activity on the concentration of pineapple residue extract (a mix of pineapple pulp, seeds, and peels), achieving 20% inhibition at a concentration of 100 µg/mL.Moreover, Hossain and Rahman [46] reported a high DPPH scavenging activity of 84.3% at a concentration of 100 µg/mL for methanolic pineapple extract (from ripe pineapple pulp).Afsharnezhad et al. [47] also reported a DPPH scavenging activity of 52.32% with a methanolic pineapple peel extract, which is similar to the result obtained from non-fermented pineapple peel (46.83%) at day 0.
Variations in antioxidant activity can primarily be ascribed to disparities in the phenolic content within the extracts.It has been well documented that the antioxidant capacity of phenolic compounds primarily stems from the presence of methoxy, hydroxyl, and carboxylic acid groups.These functional groups play a pivotal role in neutralizing free radicals, quenching singlet and triplet oxygen, and decomposing peroxides, which can play an important role in neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides [8].

Lipid Peroxidation Inhibition
Lipid peroxidation occurs when there is a large amount of reactive oxygen species (ROS), which can induce a cascade of reactions that cause oxidative stress in cell membranes, creating lipid radicals that can potentially damage proteins and DNA.Lipid peroxidation affects membrane fluidity, damages membrane proteins, and deactivates membrane receptors [48].
As shown in Figure 3, the results indicate that FPPE from L. rhamnosus, L. plantarum, and A. oryzae exhibits potential in mitigating lipid peroxidation (Figure 3A).For L. rhamnosus, it was observed that concentrations of 250 µg/mL and 500 µg/mL FPPE did not exert a significant effect on lipid peroxidation modulation.Conversely, a concentration of 1000 µg/mL consistently and significantly increased lipid peroxidation inhibition after 2 days of fermentation.When the interaction between microorganisms and fermentation time at 1000 µg/mL is compared (Figure 3D), results show that lipid peroxidation inhibition of pineapple peel extracts fermented by L. plantarum and L. rhamnosus was not statistically different on days 3 and 4 (p ≤ 0.05).Moreover, L. plantarum and L. rhamnosus FPPE at 1000 µg/mL concentrations had better lipid peroxidation inhibition compared to A. oryzae FPPE during all the fermentation processes.In the case of L. plantarum (Figure 3B), the highest lipid peroxidation inhibition (%) was at day 3 for L. rhamnosus (35.51%) and at days 3 and 4 for L. plantarum (33.99% and 32.38%, respectively) with 1000 µg/mL of FPPE.
Conversely, when considering lipid peroxidation inhibition with A. oryzae FPPE (Figure 3C), the peak inhibition was achieved on day 0 of fermentation at all three concentrations.Subsequently, there was a significant decrease in the percentage of inhibition.The higher decrease was observed from day 0 to day 1 with 250 µg/mL (−363.31%)and 1000 µg/mL (−96.21%) of FPPE, which could be due to the initial liberation of bioactive compounds bound in pineapple peel.
When the interaction between microorganisms and fermentation time at 1000 µg/mL is compared (Figure 3D), results show that lipid peroxidation inhibition of pineapple peel extracts fermented by L. plantarum and L. rhamnosus was not statistically different on days 3 and 4 (p ≤ 0.05).Moreover, L. plantarum and L. rhamnosus FPPE at 1000 µg/mL concentrations had better lipid peroxidation inhibition compared to A. oryzae FPPE during all the fermentation processes.
The results align with several studies that have evaluated the lipid peroxidation inhibition (%) of pineapple extracts.For instance, De Oliveira et al. [45] demonstrated the impact of a pineapple by-product extract, consisting of both pulp and peel components, at a concentration of 500 µg/mL.This extract exhibits a significant capacity to inhibit lipid peroxidation, with a 20% reduction observed after 30 min of reaction.Moreover, in vivo studies involving rats subjected to alcohol-induced oxidative stress further highlighted the efficacy of pineapple peel extract in mitigating lipid peroxidation.Treatment with pineapple peel extract at a dosage of 2.5 mL/kg bw (body weight) resulted in a remarkable 60.16% reduction in malondialdehyde (MDA) levels-an important biomarker of lipid peroxidation [49].Similarly, another in vivo study focused on the impact of pineapple peel extract on total phospholipids and lipid peroxidation in rat brain tissues and demonstrated that treatment with pineapple peel extract at 2.5 mL/kg bw led to a significant (72.50%) reduction in MDA levels [50].
As demonstrated in this experiment, solid-state fermentation processes have the potential to release compounds responsible for inhibiting lipid peroxidation.Variations in lipid peroxidation inhibition (%) between FPPEs derived from different microorganisms and fermentation days may be attributed to the liberation of distinct phenolic compounds during each fermentation period.
Subsequent assays did not involve the assessment of A. oryzae FPPE due to its low performance, characterized by low DPPH scavenging activity (%) and limited inhibition of lipid peroxidation (%), as observed in the preliminary assays.Consequently, the subsequent experiments exclusively employed L. rhamnosus and L. plantarum FPPE to investigate cellular antioxidant capacity, cellular anti-inflammatory potential, COX-2 production, IL-10 production, TNF-α production, IL-1β production, and IL-6 production.

Cellular Antioxidant Capacity
The cellular antioxidant capacity assay is a valuable tool for measuring the antioxidant activity of bioactive compounds in different cell cultures [51,52].The cellular antioxidant activity of fermented pineapple peel extract (25 µg/mL) by L. rhamnosus and L. plantarum in Caco2 cells is shown in Figure 4.
While cellular antioxidant activity did not show significant differences between microorganisms during fermentation with L. rhamnosus and L. plantarum, the duration of the fermentation process positively influenced cellular antioxidant activity.There was a significant and constant increase in cellular antioxidant activity observed with FPPE from day 2 to day 5 with L. rhamnosus and L. plantarum when compared to day 0. tion, IL-10 production, TNF-α production, IL-1β production, and IL-6 production.

Cellular Antioxidant Capacity
The cellular antioxidant capacity assay is a valuable tool for measuring the antioxidant activity of bioactive compounds in different cell cultures [51,52].The cellular antioxidant activity of fermented pineapple peel extract (25 µg/mL) by L. rhamnosus and L. plantarum in Caco2 cells is shown in Figure 4.While cellular antioxidant activity did not show significant differences between microorganisms during fermentation with L. rhamnosus and L. plantarum, the duration of the fermentation process positively influenced cellular antioxidant activity.There was a significant and constant increase in cellular antioxidant activity observed with FPPE from day 2 to day 5 with L. rhamnosus and L. plantarum when compared to day 0.
The highest cellular antioxidant capacity was achieved with FPPE after 5 days of fermentation with both microorganisms, resulting in a statistically significant increase (p ≤ 0.05) of 73.91% and 69.56% with L. rhamnosus and L. plantarum, respectively, compared to day 0.
The results obtained from the cellular antioxidant activity assay of FPPE show similar behavior to the DPPH scavenging activity assay.Since fermentation can transform phenolic compounds into simpler and smaller molecules, their permeability to the cell is enhanced [53].The cellular antioxidant capacity of FPPE in later days can be correlated to The highest cellular antioxidant capacity was achieved with FPPE after 5 days of fermentation with both microorganisms, resulting in a statistically significant increase (p ≤ 0.05) of 73.91% and 69.56% with L. rhamnosus and L. plantarum, respectively, compared to day 0.
The results obtained from the cellular antioxidant activity assay of FPPE show similar behavior to the DPPH scavenging activity assay.Since fermentation can transform phenolic compounds into simpler and smaller molecules, their permeability to the cell is enhanced [53].The cellular antioxidant capacity of FPPE in later days can be correlated to the amount of ferulic acid that is liberated after the solid-state fermentation of pineapple peels [54,55].
In agreement with these results, the influence of ferulic acid on cellular antioxidant activity has been previously reported.For example, Gaxiola-Cuevas et al. [56] demonstrated that a phenolic compound extract from maize tortillas, mainly composed of ferulic acid, had a cellular antioxidant capacity of 72.8% to 77.5% compared to untreated cells.Moreover, another study revealed that ferulic acid obtained from the solid-state fermentation process of wheat bran had a cellular antioxidant capacity of 63.63% compared to AAPH-induced non-treated cells.This may be explained because ferulic acid inhibits free radical-generating enzymes and improves cell protective activity [57].

Cellular Anti-Inflammatory Potential
The effects of fermented pineapple peel extract (25 µg/mL) by L. rhamnosus and L. plantarum on nitric oxide (NO) production and COX-2 protein expression in activated macrophages were tested.
As seen in Figure 5A, Raw 264.7 cells show a significant decrease in NO production in pineapple peel extracts from day 3 of fermentation by L. rhamnosus (25.5%) and day 4 by L. plantarum (47.1%), compared to day 0, respectively.FPPE at day 0 (CS) shows no significant difference in the production of COX-2 (6617.33 pg/mg protein) compared to the untreated control.This aligns with findings from Moraes et al. [62], who evaluated COX-2 inhibition in LPS-induced Raw 264.7 cells using pineapple extracts at a concentration of 50 µg/mL.These results suggest that solid-state fermentation releases anti-inflammatory compounds capable of inhibiting COX-2 production.
A greater reduction in the production of COX-2 could be partly attributed to the higher ferulic acid content in L. rhamnosus FPPE on day 5. Mir et al. [63] demonstrated that ferulic acid can protect against LPS-induced acute kidney injury in Balb/c mice, achieving COX-2 inhibition with concentrations of 100 mg/kg and 50 mg/kg.
In another study, the inhibitory potential of Terminalia bellirica (Gaertn) Roxb fruit extracts was assessed in LPS-induced Raw 264.7 cells, showing a significant COX-2 inhibition attributed to its phenolic compound composition.Notably, one of the main compounds of this fruit was ferulic acid [64].Furthermore, Villela-Castrejón et al. [53] demonstrated that spray-dried nejayote, rich in ferulic acid (constituting 77.8% of the polyphenols) after in vitro digestion, exhibited an inhibitory effect on COX-2 in LPS-induced Raw 264.7 cells.
These results indicate that the inhibitory effect of FPPE on NO production might be responsible for suppressing COX-2 protein expression production in cells stimulated with LPS.Additionally, the observed reduction in anti-inflammatory cytokines may be Although fermentation was not significantly different between microorganisms, the fermentation process positively affected nitric oxide production.The treatments that had the lowest nitric oxide production compared to FPPE at day 0 (88.36%) were L. rhamnosus (38%) and L. plantarum (46.7%)FPPE at day 4 (p ≤ 0.05).
It was observed that there was a significant increase in NO production with L. rhamnosus (59.9%) and L. plantarum (40.0%)FPPE at day 5 (p ≤ 0.05) compared to day 4.This increase can be attributed to the higher concentration of phenolic compounds, including ferulic acid, which has been associated with pro-inflammatory activity at elevated concentrations [58][59][60].The enzymes produced by L. rhamnosus and L. plantarum during fermentation hydrolyze ferulic acid glyceride bonds, resulting in the availability of phenolic acids that enhance their anti-inflammatory properties [53].
A study by Pongjanta and Chansiw [61] showed that their non-fermented pineapple peel extract, derived from two pineapple varieties from Thailand, exhibited NO production levels of 77.28%, 71.81%, and 64.47% for the Nanglae variety and 66.73%, 65.35%, and 63.67% for the Phulae variety at extract concentrations of 10 µg/mL, 100 µg/mL, and 1000 µg/mL, respectively.NO production in this study shows slight changes even at higher concentrations (1000 µg/mL).Differences between these findings and previous reports could be attributed to variations in plant variety and maturity stage.
The effects of FPPE on COX-2 protein expression were detected by ELISA analysis.Results demonstrate that L. rhamnosus and L. plantarum FPPE at 25 µg/mL concentrations reduced the production of COX-2 (>59.07%)(Figure 5B).
Moreover, when comparing COX-2 production between 4LR and 5LR, there were no statistically significant differences between these two treatments (p ≤ 0.05).Likewise, there was no statistically significant difference between treatments with L. plantarum FPPE on day 4 (3555.44pg/mg protein) and day 5 (3717 pg/mg protein) (p ≤ 0.05).
FPPE at day 0 (CS) shows no significant difference in the production of COX-2 (6617.33 pg/mg protein) compared to the untreated control.This aligns with findings from Moraes et al. [62], who evaluated COX-2 inhibition in LPS-induced Raw 264.7 cells using pineapple extracts at a concentration of 50 µg/mL.These results suggest that solidstate fermentation releases anti-inflammatory compounds capable of inhibiting COX-2 production.
A greater reduction in the production of COX-2 could be partly attributed to the higher ferulic acid content in L. rhamnosus FPPE on day 5. Mir et al. [63] demonstrated that ferulic acid can protect against LPS-induced acute kidney injury in Balb/c mice, achieving COX-2 inhibition with concentrations of 100 mg/kg and 50 mg/kg.
In another study, the inhibitory potential of Terminalia bellirica (Gaertn) Roxb fruit extracts was assessed in LPS-induced Raw 264.7 cells, showing a significant COX-2 inhibition attributed to its phenolic compound composition.Notably, one of the main compounds of this fruit was ferulic acid [64].Furthermore, Villela-Castrejón et al. [53] demonstrated that spray-dried nejayote, rich in ferulic acid (constituting 77.8% of the polyphenols) after in vitro digestion, exhibited an inhibitory effect on COX-2 in LPS-induced Raw 264.7 cells.
These results indicate that the inhibitory effect of FPPE on NO production might be responsible for suppressing COX-2 protein expression production in cells stimulated with LPS.Additionally, the observed reduction in anti-inflammatory cytokines may be attributed to the ability of the present phenolic compounds to inhibit nuclear factor-κB (NF-κB), a pivotal transcription factor responsible for the activation of genes related to inflammatory mediators, including inducible nitric oxide synthase (iNOS) and cyclooxygenases (COXs) [23].

Cytokines Production
Because inflammation is a complex system where cytokines are involved, it is important to measure their activity.The influence of pineapple peel extracts fermented by L. rhamnosus and L. plantarum on the release of anti-inflammatory cytokine IL-10 (interleukin 10), and pro-inflammatory cytokines IL-6 (interleukin 6), IL-2 (interleukin 2), IL-1β (interleukin 1β), and TNF-α (tumor necrosis factor-alpha) was evaluated.The results are shown in Figure 6.
L. rhamnosus FPPE at day 5 (5LR) reduced the production of IL-2, IL-6, IL-1β, and TNFα (Figure 6) and increased the production of IL-10 in LPS-treated Raw 264.7 cells (Figure 7).There was a statistically significant difference between all the treatments compared to the untreated control (p ≤ 0.05).
Treatments with 5LR and CS show a significant decrease in IL-2 production by 32.61% and 26.67%, compared to control, respectively.A statistically significant difference was observed between these two treatments (p = 0.0009).Furthermore, L. rhamnosus FPPE on day 5 (5LR) exhibits a similar effect to that of Indomethacin (1 µg/mL) and Dexamethasone (1 µg/mL), two reference anti-inflammatory agents, in reducing IL-2 production (32.06% and 34.45%, respectively).A significant reduction in IL-6 production was observed with the 5LR treatment (−22.78%) compared to the untreated control.However, both Indomethacin (−60.56%) and Dexamethasone (−68.95%)demonstrate a higher reduction compared to the control.Notably, the CS treatment exhibits no statistically significant difference when compared to the untreated control (p ≤ 0.05) (Figure 6B).These findings align with those reported by Ajayi et al. [65] who noted a 27.27% reduction in IL-6 production in rats with high-fat dietinduced memory impairment and anxiety-like behavior after receiving a pineapple peel extract (200 mg/kg) compared to untreated rats.
A significant decrease of 39.6% in IL-1β production (Figure 6C) was observed with L. rhamnosus FPPE on day 5 (5LR) compared to the untreated control (p ≤ 0.05).No statistically significant differences were observed between treatments.
In addition, treatments with 5LR and CS show a significant decrease in TNF-α production (Figure 6D) by 67.34% and 60.22%, compared to control, respectively.A significant reduction in IL-6 production was observed with the 5LR treatment (−22.78%) compared to the untreated control.However, both Indomethacin (−60.56%) and Dexamethasone (−68.95%)demonstrate a higher reduction compared to the control.Notably, the CS treatment exhibits no statistically significant difference when compared to the untreated control (p ≤ 0.05) (Figure 6B).These findings align with those reported by Ajayi et al. [65] who noted a 27.27% reduction in IL-6 production in rats with high-fat diet-induced memory impairment and anxiety-like behavior after receiving a pineapple peel extract (200 mg/kg) compared to untreated rats.
A significant decrease of 39.6% in IL-1β production (Figure 6C) was observed with L. rhamnosus FPPE on day 5 (5LR) compared to the untreated control (p ≤ 0.05).No statistically significant differences were observed between treatments.
In addition, treatments with 5LR and CS show a significant decrease in TNF-α production (Figure 6D) by 67.34% and 60.22%, compared to control, respectively.

Conclusions
This study demonstrates that solid-state fermentation of pineapple peels using lactic acid bacteria (L.plantarum and L. rhamnosus) and filamentous fungi (A.oryzae) leads to an increased release of phenolic compounds that are typically bound within the pineapple peel matrix.Furthermore, this enhanced release of phenolic compounds also increased their potential as antioxidants and anti-inflammatory agents.The anti-inflammatory activity was correlated to the significant inhibition of pro-inflammatory cytokines, including COX-2, IL-2, IL-6, IL-1β, and TNF-α, and the promotion of IL-10 production, which is known for its potent anti-inflammatory properties.
Remarkably, the profiles of phenolic compounds from pineapple peel were found to vary depending on the microorganism used for solid-state fermentation.Consequently, different microorganisms can be selected based on the intended application.For instance, for higher total phenolic compound (TPC) production, solid-state fermentation with L. plantarum over 5 days is recommended.Conversely, for greater nitric oxide (NO) inhibition, solid-state fermentation with L. rhamnosus for 4 days is more suitable.Likewise, L. plantarum and L. rhamnosus produced a significant amount of ferulic acid and vanillin, compounds commonly used in the food and cosmetic industries.In contrast, A. oryzae generates furanones, which find widespread application in the food industry, imparting specific aromas to food products such as the pineapple scent.
Since a great variety of value-added compounds can be produced using pineapple waste, it is essential to thoroughly analyze its production yields and scalability in the industrial field.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1,Table S1: Chemical composition of pineapple peel; Figure S1: pH and Aw changes during solid-state fermentation of pineapple peels with L. rhamnosus, L. plantarum, and A. oryzae.

Conclusions
This study demonstrates that solid-state fermentation of pineapple peels using lactic acid bacteria (L.plantarum and L. rhamnosus) and filamentous fungi (A.oryzae) leads to an increased release of phenolic compounds that are typically bound within the pineapple peel matrix.Furthermore, this enhanced release of phenolic compounds also increased their potential as antioxidants and anti-inflammatory agents.The anti-inflammatory activity was correlated to the significant inhibition of pro-inflammatory cytokines, including COX-2, IL-2, IL-6, IL-1β, and TNF-α, and the promotion of IL-10 production, which is known for its potent anti-inflammatory properties.
Remarkably, the profiles of phenolic compounds from pineapple peel were found to vary depending on the microorganism used for solid-state fermentation.Consequently, different microorganisms can be selected based on the intended application.For instance, for higher total phenolic compound (TPC) production, solid-state fermentation with L. plantarum over 5 days is recommended.Conversely, for greater nitric oxide (NO) inhibition, solid-state fermentation with L. rhamnosus for 4 days is more suitable.Likewise, L. plantarum and L. rhamnosus produced a significant amount of ferulic acid and vanillin, compounds commonly used in the food and cosmetic industries.In contrast, A. oryzae generates furanones, which find widespread application in the food industry, imparting specific aromas to food products such as the pineapple scent.
Since a great variety of value-added compounds can be produced using pineapple waste, it is essential to thoroughly analyze its production yields and scalability in the industrial field.

Figure 1 .
Figure 1.Concentration of total phenolic compounds in pineapple waste fermented for 5 days by L. rhamnosus (LR), L. plantarum (LP), and A. oryzae (AO).Values represent the mean of three replicates with their standard error bars.Different letters among bars indicate statistical differences in the content of total phenolic compounds (TPC) between fermentation times (days) using the Tukey test (p < 0.05).Abbreviations: total phenolic compounds (TPC), gallic acid equivalents (GAE), fermented pineapple peel extract (FPPE).

Figure 1 .
Figure 1.Concentration of total phenolic compounds in pineapple waste fermented for 5 days by L. rhamnosus (LR), L. plantarum (LP), and A. oryzae (AO).Values represent the mean of three replicates with their standard error bars.Different letters among bars indicate statistical differences in the content of total phenolic compounds (TPC) between fermentation times (days) using the Tukey test (p < 0.05).Abbreviations: total phenolic compounds (TPC), gallic acid equivalents (GAE), fermented pineapple peel extract (FPPE).

Figure 4 .
Figure 4. Cellular antioxidant capacity of fermented pineapple peel extracts (25 µg/mL) by L. rhamnosus (LR) and L. plantarum (LP) in Caco2 cells.Values represent the mean of three replicates with their standard error bars.Different letters among bars indicate statistical differences in the cellular antioxidant capacity between microorganisms and fermentation time (days) using the Tukey test (p < 0.05).

Figure 4 .
Figure 4. Cellular antioxidant capacity of fermented pineapple peel extracts (25 µg/mL) by L. rhamnosus (LR) and L. plantarum (LP) in Caco2 cells.Values represent the mean of three replicates with their standard error bars.Different letters among bars indicate statistical differences in the cellular antioxidant capacity between microorganisms and fermentation time (days) using the Tukey test (p < 0.05).

Table 1 .
Identification of bioactive compounds obtained from the fermented pineapple peel extracts (FPPE).

Table 1 .
Identification of bioactive compounds obtained from the fermented pineapple peel extracts (FPPE).

Table 2 .
Cont.Concentrations are reported for each individual standard.Compounds were quantified at 280, 320, and 360 nm.Concentrations are expressed as equivalents of gallic acid, p-coumaric acid, ferulic acid, and quercetin.2Valuesrepresent the mean of three replicates ± standard error of the mean.
3Different letters in the same column indicate statistical differences in the concentration of each compound between treatments using the least significant difference (LSD) test (p < 0.05).Abbreviations: LR: L. rhamnosus; LP: L. plantarum; AO: A. oryzae; N.I.: No identified; ND: Not detectable.