Valorization of Coffee Cherry By-Products Through Fermentation by Human Intestinal Lactobacilli in Functional Fermented Milk Beverages

Academic Editor: José António Couto Received: 28 November 2024 Revised: 10 December 2024 Accepted: 20 December 2024 Published: 27 December 2024 Citation: Picon, A.; Campanero, Y.; Sánchez, C.; Álvarez, I.; RodríguezMínguez, E. Valorization of Coffee Cherry By-Products Through Fermentation by Human Intestinal Lactobacilli in Functional Fermented Milk Beverages. Foods 2025,14, 44. https://doi.org/10.3390/ foods14010044 Copyright: © 2024 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/). Article Valorization of Coffee Cherry By-Products Through Fermentation by Human Intestinal Lactobacilli in Functional Fermented Milk Beverages Antonia Picon 1,* , Yolanda Campanero 1, Carmen Sánchez 1, Inmaculada Álvarez 2and Eva Rodríguez-Mínguez 1 1 Departamento de Tecnología de Alimentos, INIA, CSIC, Carretera de La Coruña Km 7, 28040 Madrid, Spain 2Unidad de Servicio de Técnicas Analíticas, ICTAN, CSIC, Calle JoséAntonio Novais 6, 28040 Madrid, Spain *Correspondence: [email protected] Abstract: During coffee production, the removal and disposal of the coffee beansurrounding layers pose an environmental problem. In this work, we examined the effects of several aqueous coffee cherry extracts on the growth and metabolism, biofilm formation, antioxidant capacity and antimicrobial activity of six lactobacilli from the INIA collection and a commercial probiotic Lactobacillus rhamnosus GG strain. Growth medium supplementation with different coffee cherry extracts (at 40%) stimulated strain growth and metabolism. The ground cherry pulp extract (CPE) with the highest total polyphenol content was selected for further use. This CPE contained alkaloids, phenolic acids and flavonoids. Upon CPE supplementation, some strains significantly (p< 0.01) increased biofilm formation, while all strains increased antioxidant capacity and antimicrobial activity. After preliminary tests, we developed three bifunctional dairy products, containing 20% CPE and fermented with strains INIA P495, INIA P708 or GG. These strains maintained high levels after manufacture, refrigerated storage, and throughout an in vitro procedure mimicking gastrointestinal tract conditions. Compared to controls, CPE-containing products showed increased levels of total polyphenol compounds, antioxidant capacity and antimicrobial activity, together with positive sensory characteristics. CPE and these selected strains could thus be used to elaborate innovative functional fermented milk products. Keywords: coffee cherry pulp; lactobacilli; antioxidant capacity; antimicrobial activity; fermented milk; starters; fermented foods; functional foods 1. Introduction Coffee is one of the most widely consumed beverages in the world. The estimated total world coffee production in 2022 was 10.8 million tons [ 1 ]. The coffee sector is still growing due to increasing consumption in emerging economies, a strong interest in specialty coffee and product innovations in developed countries [ 2 ]. The two major coffee species consumed worldwide are Coffea arabica (Arabica) and Coffea canephora (Robusta). Arabica coffee is considered to be superior to Robusta due to its organoleptic properties [ 3 ]. Interest in C. canefora has been steadily growing, however, due to its considerable resistance to climate change [4]. Relevant factors determining the coffee quality include agricultural procedures (mostly genetic makeup of the beans and their ripeness, cultivation and harvesting methods, seasonality, altitude and climate), processing methods, bean roasting and grinding, as well as beverage preparation and consumption [ 5 ]. Since coffee is produced from roasted Foods 2025,14, 44 https://doi.org/10.3390/foods14010044 Foods 2025,14, 44 2 of 18 beans, the surrounding layers (silver skin, parchment, mucilage, pulp and skin) have to be removed and are discarded as waste [ 6 ]. Unsafe disposal of the corresponding waste has a negative impact on the environment in coffee-producing countries, due to high acidity and caffeine, polyphenol and tannin concentrations [ 7 ]. To improve the sustainability of coffee production, bioactive compounds such as alkaloids, melanoidins and polyphenols could be obtained from these by-products and used for the development of nutraceuticals, functional foods or cosmetics [ 6 ]. Several reviews dealing with different aspect of coffee by-product valorization have been recently published, providing compositional data, their incorporation as ingredients in food formulations and new product development [ 8 ], application of fermentation in the coffee industry [ 9 ], and as prebiotic ingredients [ 10 ]. These reviews highlight the great industrial potential of coffee by-products. Polyphenol compounds are secondary plant metabolites with several protective roles for humans. They may act locally against oxidative damage at the gastrointestinal tract (GIT), and systemically by reducing the risk of several chronic diseases [ 11 ]. Polyphenols are classified as prebiotics, i.e., substrates that are selectively utilized by host microorganisms conferring health benefits by the International Scientific Association for Probiotics and Prebiotics [ 12 ]. Therefore, dairy product health benefits could be further improved by supplementation with polyphenol-rich extracts [13]. Coffee microbiota mostly comprise bacteria (lactobacilli, Bacillus,Arthrobacter,Acinetobacter,Klebsiella and Weissella) and yeast (Saccharomyces,Pichia,Candida,Rhodotorula, Hanseniaspora and Kluyveromyces) [ 14 ]. During coffee processing, coffee microbiota plays a pivotal role in fermentation, significantly impacting the coffee’s chemical composition and sensory attributes [5]. Moreover, lactobacilli have a prominent role in fermented dairy products [ 15 ]. They are recognized as human gut microbiota commensals and have been used as probiotics for a long time [ 15 ]. Lactobacilli are able to transform complex dietary polyphenol compounds, increasing their bioavailability [ 16 ]. Depending on individual microbiota composition, however, great variations in polyphenol transformation have been observed [17]. Consumers with limited endogenous polyphenol-transforming abilities in particular could benefit from the supplementation of dairy products with polyphenol-rich extracts. Fermentation with transformation-capable intestinal lactobacilli strains could thus provide a cost-effective method to reach a wide public. The aims of this work were: (1) to examine the effects of aqueous coffee cherry extracts on several lactobacilli strains (most of them from intestinal origin), and (2) to develop bifunctional dairy products containing a coffee cherry pulp extract (CPE) and one of the strains able to increase its polyphenol bioavailability. 2. Materials and Methods 2.1. Coffee Cherry Extract Preparation and Preliminary Characterization The coffee (Coffea canephora) cherries used in this work were collected and sun-dried by local farmers for Caféau Light Group S.L. (Madrid, Spain) in Guinea Conakry during the 2022 harvest season. After air transport, washing and drying, they were vacuum packaged and stored at –30 ◦C until processing. After thawing, pulp and bean were mechanically separated. Part of the pulp was ground in a coffee grinder (Jata, Model ML132, Tudela (Navarra), Spain) to a homogeneous powder with a particle size of less than 2 mm. Four types of extraction were performed, each with 10 volumes of sterile distilled water (in a similar way to preparing an herbal tea). For the first two, whole pulp or ground pulp were mixed with boiling water and kept at room temperature for 7 min. For the third method, ground pulp was mixed with sterile water at 4 ◦ C and kept at this temperature for 30 min. For the last method, beans were mixed Foods 2025,14, 44 3 of 18 with boiling water and kept at room temperature for 7 min. Extracts were passed through a stainless-steel strainer and filtered through a 0.22 µ m Cytiva Whatman GD/X filter (Global Life Science Solutions Operations UK Ltd., Little Chalfont, Buckinghamshire, UK). To test extract sterility, in duplicate, 50 µ L portions of each extract were spread on plate count agar (PCA, Difco, Beckton, Dickinson & Co, Le Pont de Claix, Francia) and yeast glucose chloramphenicol agar (YGC, Difco, Beckton, Dickinson & Co, Le Pont de Claix, Francia) plates. Incubations were performed at 30 ◦ C for 48 h for PCA plates and at 25 ◦C for five days for YCG plates. Extract pH and absorbance at 600 nm (A 600 , as an indication to color differences) were measured, in triplicate, with a Beckman DU 650 spectrophotometer (Beckman Instruments S.A., Madrid, Spain) and a Crison pH-meter (model Basic 20, Crison Instruments, Barcelona, Spain), respectively. Following the method of Velioglu et al. [ 18 ], we used gallic acid and the Folin-Ciocalteau reagent (both from Sigma-Aldrich Co., St. Louis, MI, USA.) to determine, in triplicate, the total polyphenol content (TPC). The results are represented as µ g of gallic acid equivalents (GAE) per mL. 2.2. Strains Used and Growing Conditions Six strains, part of the INIA culture collection, were used in this work. Five strains, Lacticaseibacillus (Ls.) paracasei ssp. paracasei INIA P495 and INIA P708, Ls. rhamnosus INIA P334, Lactobacillus gasseri INIA P508, and Limosilactobacillus mucosae INIA P459 originated from breast-fed infants [ 19 ]. These five strains survived the major GIT conditions well and showed a broad inhibitory activity and limited coaggregation with bacterial pathogens. Also, these strains were susceptible to beta-lactamase inhibitors and protein synthesis inhibitors while not bearing mucinolytic activity or producing biogenic amines [ 19 ]. Strain INIA P459 possessed bile salt hydrolase activity and produced hydrogen peroxide [ 19 ]. Strain INIA P708 possessed a ropy phenotype and produced exopolysaccharides (EPS). The sixth strain, Lactiplantibacillus (Lp.) plantarum ssp. plantarum INIA TAB84, was a dairy isolate that produces an uncharacterized plantaricin [ 20 ]. As reference, the commercial probiotic Ls. rhamnosus GG strain [21] was included in the study. Strains were anaerobically (Anaerogen, Oxoid, Basingstoke, UK) grown at 37 ◦ C for 24 h on plates of de Man, Rogosa and Sharpe medium (MRS, Difco) with 0.05% cysteine-HCl (Sigma-Aldrich Co.) (MRSC) and 1.5% agar (Difco). 2.3. Effect of Coffee Cherry Extracts on Lactobacilli Metabolism To test metabolic activity, we followed the procedure developed in our previous study [ 22 ]. Briefly, brain heart infusion (BHI, Oxoid) medium with 0.05% cysteine-HCl, 0.03% methyl blue (Sigma-Aldrich Co.) and 1.5% agar (Difco) was dissolved in 60% of the total water volume and sterilized. One of the extracts, or water (control plates), was added aseptically to make up the total volume. Solidified plates were stored at 4 ◦C until use. Drops (5 µ L) of bacterial cell suspensions (in sterile 0.2% peptone water with 0.05% cysteine-HCl, at approximately 8.5 log CFU/mL) were placed on plates containing individual coffee cherry extracts or no extract (control plates), in duplicate. After anaerobic incubation at 37 ◦ C for 24 h, bacterial growth and dye color alteration were recorded, following the code described in [ 22 ]: 0 = no growth, 1 = faint growth without color change, 2 = growth and dye change to blue, 3 = growth and a blue ring of up to 2 mm around the drop and 4 = growth and a blue ring of more than 2 mm around the drop. 2.4. Preparation of the Coffee Cherry Pulp Extract (CPE) The coffee cherry pulp was placed in a kitchen robot (Thermomix, model TM31, Vorwerk España M.S.L., S.C., Madrid, Spain) and subjected to blade speeds of 6 for 60 s and 8 for 30 s. Approximately 70 g were weighed in each of the two glass beakers, mixed with Foods 2025,14, 44 4 of 18 10 volumes of boiling water and incubated for 7 min in a stove at 50 ◦ C (Memmert GmbH, Schwabach, Germany). The mixture was briefly stirred, and particulate material was removed by filtering through a sterile gauze. Microbial sterility was achieved by vacuum filtration through a 0.2 µ m polyethersulfone membrane (TPP Techno Plastic Products AG, Trasadingen, Switzerland). Sterile 50 mL amber plastic tubes (Eppendorf AG, Hamburg, Germany) were used to store extract aliquots at –40 ◦C. 2.5. CPE Characterization Extract sterility, pH, TPC and A 600 were determined as described in Section 2.1. CPE dry matter was determined as percentage of the initial weight. The total sugar content was measured by the colorimetric phenol-sulfuric acid method [23] using glucose (SigmaAldrich Co.) as a standard, and protein content was assayed with a Coomasie Plus (Bradford) reagent kit (Thermo Scientific, Rockford, IL, USA) and bovine serum albumin (BSA) as standard [ 22 ]. The Benzie & Strain method [ 24 ], with modifications and Trolox (Sigma Aldrich Co.) as standard, was followed to determine the CPE antioxidant capacity [ 22 ]. All determinations in this section were performed in triplicate. In order to identify polyphenol compounds, the CPE was subjected to HPLC-PAD/ HPLC-ESI-MS, at the ICTAN (CSIC) Analysis Service Unit, following the method and equipment previously described [22]. Analyses were carried out in duplicate. 2.6. CPE Effect on Lactobacilli Growth and Metabolism Growth experiments were performed in BHI broth with 0.05% cysteine-HCl (BHIC) to analyze, in a quantitative manner, the effect of the CPE on strain growth and survival. BHIC broth was supplemented with 40% CPE, while broth without CPE served as control. Strains were inoculated in broth at an approximate level of 7.5 log CFU / mL and incubated under anaerobic conditions at 37 ◦ C for 6 days. Microbial levels, pH and total polyphenol content were determined at days 1 and 6. 2.7. CPE Effect on Lactobacilli Biofilm Formation To study the effect of CPE on biofilms, we followed the method of Lee et al. [ 25 ], with some modifications [ 22 ]. Briefly, 200 µ L of BHIC broth containing 0.02% oxgall (Sigma-Aldrich Co.) and CPE or water (control) were placed in each well of 96-multiwell plates (Thermo Fisher Scientific, Roskilde, Denmark) and inoculated with 2 µ L of bacterial suspensions (prepared as described in Section 2.3). For each strain, twelve replicates, divided over two independent experiments, were prepared. Non-inoculated wells were included as negative controls. After 24 h of anaerobic incubation at 37 ◦ C, the liquid phase was discarded, and the wells stained with 0.4% crystal violet solution for 30 minutes. After washing thrice with distilled water, biofilms were resuspended in 30% glacial acetic acid and quantified by reading A 600 in a microplate reader (Multiskan Spectrum, Thermo Fisher Scientific Oy, Vantaa, Finland). 2.8. CPE Effect on Lactobacilli Antioxidant Capacities The antioxidant capacity of the strains was evaluated by measuring the reduction of the 2,2-diphenyl-1-picrylhydracyl (DPPH, Sigma-Aldrich Co.) radical [ 26 ]. Strains, grown on BHIC plates, supplemented or not with 40% CPE, were suspended in 0.85% NaCl and adjusted to an OD 600 of 1.2. In triplicate, cell suspensions (400 µ L) were mixed with DPPH 0.2 M (500 µ L) and incubated protected from light at room temperature for 30 min. After centrifugation at 6026 g for 3 min, A 517 was measured. A 0.85% NaCl solution (400 µ L) was used as blank. Total antioxidant capacity (TAC) of each culture was calculated as the percentage of DPPH reduction: TAC = [1 −(A517 (sample)/A517 (blank))] ×100. Foods 2025,14, 44 5 of 18 2.9. CPE Effect on Lactobacilli Antimicrobial Activities CPE antimicrobial activity toward four pathogenic strains of the INIA collection was studied, in a similar way as previously described [ 22 ]. Gram-positive strains Clostridium perfringens CECT 486 (C) and Listeria monocytogenes Scott A CECT 5672 (L) and Gramnegative strains Klebsiella oxytoca INIA col 108 (K) and Salmonella enterica serovar Enteritidis CECT 4155 (S) were tested. Strains were grown in BHI at 37 ◦ C for 24 h under anaerobic conditions (Clostridium perfringens CECT 486) or as stand cultures. The stand-alone inhibitory activity of the CPE on pathogenic strains was evaluated following the method previously described [ 22 ]. First, 5 mL of 50 ◦ C-tempered 0.5% BHI soft agar, previously inoculated with fresh cultures of each pathogenic strain at a 0.1% (final concentration, approx. 5.5–6 log CFU/mL), were layered on top of solidified BHI plates, in duplicate. Next, 5 mm-diameter wells were bored with a sterile punch. Finally, each well was filled with the serially diluted CPE extract (100%, 50%, 40%, 20%, 10%, 5%, 1%, 0.5%, 0.25%, 0.125% and 0.0625%; 50 µL per well). Plates were incubated at 37 ◦C for 24 h. To study the combined antimicrobial activity of the selected strains (four intestinal strains plus the GG reference strain) and CPE against pathogenic strains, suspensions of cells grown on BHIC (control) or BHIC + 40% CPE plates were prepared as described in Section 2.3. Drops (5 µ L) of each suspension were deposited on a new BHI agar plate. After anaerobic incubation at 37 ◦ C for 24 h, a layer of a 50 ◦ C-tempered inoculated BHI soft agar, previously inoculated with fresh cultures of each pathogenic strain (prepared as explained above), was poured on top. Plates were examined for the presence of inhibition halos after 24 h incubation at 37 ◦C. 2.10. Manufacture of CPE Fermented Milk Products Before starting manufacturing, a group (n = 8) of trained panelists performed a preliminary test to choose a extract concentration suitable for sensory evaluation. UHT milk (Pascual, Burgos, Spain) samples supplemented with 20 or 40% CPE extracts were tested. For the manufacture, two independent experiments were performed. UHT milk (Pascual, Burgos, Spain) was supplemented with 20% CPE or sterile water (control) and distributed in 15 ml sterile polypropylene tubes (VWR, Radnor, PA, USA). Control and CPE-supplemented milk tubes were inoculated at 1% with each cell suspension (prepared as in Section 2.3) of the two selected strains (Ls. paracasei subsp. paracasei INIA P495 and INIA P708) and the reference strain, Ls. rhamnosus GG, at a final concentration of approximately 7.5 log CFU/mL. Non-inoculated control tubes were included. All tubes were anaerobically incubated at 37 ◦ C for 24 h. Fermented milk products were introduced in a cold chamber (4 ◦C) and stored for up to 20 days. 2.11. CPE Fermented Milk Product Characterization Fermented milk products were analyzed after 1, 12 and 20 days. Microbial levels were determined in duplicate, as described in Section 2.2. To test strain survival in vitro under major GIT conditions, the method developed by Langa et al. [ 27 ] was applied. Briefly, fermented dairy product samples (1 mL) were mixed with a pH 3.0 buffered PBS solution ( 9 mL ) at 37 ◦ C for 90 s. Mixtures were anaerobically incubated at 37 ◦ C for 1 h. Thereafter, mix samples (1 mL) were added to 9 mL of bile solution (0.15%, ox-bile desiccated; Oxoid) and kept anaerobically at 37 ◦ C for 1 h, before microbial enumeration. Product antimicrobial activities were tested as described in Section 2.9. Fermented dairy product pH values were determined in triplicate. Following the method of del Olmo et al. [ 28 ], polyphenol compounds were extracted. Briefly, samples (1 mL) were subjected to a first extraction with an HCl-acidified (16 mM) methanol:water (50:50) solution (9 mL), and a second one with an acetone:water (70:30) solution (9 mL). Foods 2025,14, 44 6 of 18 Both extractions were performed under constant agitation at room temperature for 1 h. Extracts were mixed to a 1:1 proportion and filtered (0.22 µ m PVDF membrane filter, Millex-GV, Merck Millipore Ltd., Cork, Ireland). Filtrates were used to determine, in triplicate, fermented dairy product TPC and antioxidant capacity, as described in Sections 2.1 and 2.5, respectively. After 24 h of refrigerated storage, a group of trained panelists (n = 8) performed a preliminary sensory test. Panelists received information about the samples, procedures and their rights. We collected their signed written consent to participate. For the descriptive test, a specific sensory lexicon was developed [ 29 ], and thereafter, panelists were asked to describe aspect, odor and flavor characteristics. 2.12. Statistical Analysis Two-way analysis of variance (ANOVA) with presence/absence of CPE and strain as main effects (SPSS 25.0 statistical package, IBM Corporation, Armonk, NY, USA) was applied to data. Tukey’s test by one-way ANOVA, with significance assigned at p< 0.01, was then performed to compare means (presence/absence of CPE or strain). 3. Results and Discussion 3.1. Coffee Cherry Extracts: Preliminary Characterization and Selection Four coffee cherry extracts, three from pulp and one from bean, were obtained and evaluated (Table 1). Pulp extracts displayed significantly (p< 0.01) lower pH, A 600 and TPC values than bean extract. Among the extracts, the ground pulp prepared with boiling water showed the highest TPC. Table 1. Preliminary characterization of the coffee cherry extracts 1. Variable Extracts Whole Pulp Ground Pulp Ground Pulp 4 ◦C Bean + Silverskin pH 3.78 ±0.02 b3.76 ±0.01 b3.74 ±0.01 b5.41 ±0.01 a A600 0.0268 ±0.0100 bc 0.0460 ±0.0093 b0.0193 ±0.0102 c0.1352 ±0.0098 a TPC 2136.15 ±1.16 c314.18 ±0.61 a160.48 ±0.45 b70.36 ±0.42 d 1 Coffee cherry extracts were prepared with sterile distilled water (at a 1:10 ratio) at 95 ◦ C for 7 min, or at 4◦C for 30 min. 2 TPC = Total polyphenol content, in µ g GAE/mL. All variables were mean ± SD of triplicate determinations in two experiments (n = 6). Means with lower-case superscripts differ significantly at p< 0.01. The results on the effects of coffee cherry extracts on strain metabolism are shown in Table 2. Bacterial growth was qualitatively recorded in all plates after 24 h incubation. All strains showed limited growth and dye color change in control plates and in plates with the bean extract. Table 2. Behaviour of intestinal strains grown on BHI agar with methyl blue (control, BHIC) and supplemented with 40% of the coffee cherry extracts incubated at 37 ◦C for 48 h under anaerobic conditions. Strain Control Extract Whole Pulp Ground Pulp Ground Pulp 4 ◦C Bean + Silver Skin INIA P495 1+ 3 4 4 2 INIA P708 1+ 3 4 4 2 INIA P334 1 2 3 3 1+ INIA TAB84 1 2 3 3 1 INIA P508 1 2 3 3 1 INIA P459 1 3 3 3 1 GG 1+ 3 4 4 1+ Codes: 0 = no growth; 1 = light growth without color change; 2 = growth and change to blue color; 3 = blue ring of up to 2 mm around the drop; 4 = blue ring of more than 2 mm around the drop. Foods 2025,14, 44 7 of 18 However, the three pulp extracts stimulated strain metabolism, showing blue rings (due to acid production) of bigger diameters than in the control plates. The best scores were recorded for strains grown with ground pulp extracts, irrespective of extraction temperature. The biggest stimulations were recorded for strains INIA P495, INIA P708 and the GG reference strain. In contrast to what we observed in this work, with 40% concentrations well tolerated by all lactobacilli strains, extract concentrations of mangosteen fruit above 10% were detrimental for the growth of these strains [22]. Taking into account all these results, the ground pulp extract prepared with boiling water was selected and prepared at a large scale. 3.2. CPE Characterization The physico-chemical characteristics for this coffee cherry pulp extract are shown in Table 3. Both pH and A 600 values were similar to the ones obtained for the ground pulp extract prepared with boiling water. Sucrose is the most abundant sugar present in coffee; it has an important contribution to the taste of this beverage [ 30 ]. A raw filtrate of the wet coffee reached a sugar content of 1.67 mg GE/mL [ 31 ]. Taking into account that the ratios used in the wet processing were 1 Kg pulp:80–120 L water [ 31 ], and that we used a 1:10 ratio , the total sugar content of our CPE was lower than expected. These differences could be explained, however, by the large variations in sucrose found between diverse species and origins and even within beans from the same batch [ 30 ]. In comparison with previous data on mangosteen extracts [ 22 ], CPE had a lower sugar content than mangosteen pulp extract (12.14 mg GE/mL) or even mangosteen rind extract (9.91 mg GE/ml). Table 3. Characteristics of the coffee cherry pulp extract. pH A600 DM 1 (%) Sugar Content (mg GE/mL) Protein Content (µg BSA/mL) TPC 2 (µg GAE/mL) Antioxidant Capacity (mM TE) 3.81 ±0.01 0.0451 ±0.01 1.98 ±0.03 8.28 ±0.21 47.39 ±4.37 406.43 ±1.50 8.36 ±0.09 1 DM = dry matter and 2 TPC = Total polyphenol content. All variables were mean ± SD of triplicate determinations in two experiments (n = 6). The higher TPC of the CPE, in comparison to the ground pulp extract previously prepared, might be due to the higher extraction temperature of 50 ◦ C vs room temperature [ 32 ]. The TPC of the CPE was lower than the 1.3 mg GAE / mL obtained for the raw filtrate of wet coffee processing [ 31 ]. However, TPC on a weight basis (4.06 mg GAE/g fresh pulp) was in agreement with the 4.9 mg GAE/g pulp DM [ 33 ]. Compared to the mangosteen extracts of our previous work [ 22 ], CPE contained a much lower TPC (5.70 and 10.01 mg GAE / mL for pulp and rind, respectively). Accordingly, the CPE antioxidant capacity was lower than those of the mangosteen extracts (100.95 and 186.60 mM TE for pulp and rind, respectively). The most abundant phenolic compounds detected in the CPE by HPLC-PAD/HPLCESI-MS (Figure 1) were, in decreasing amounts (Table 4), the alkaloids trigonelline and caffeine, protocatechuic and chlorogenic acids, caffeoylquinic and feruloylquinic acids, the flavan-3-ol catechin and, in lower quantities, caffeic acid, the flavonoid glycoside rutin, several dicaffeoylquinic acid isomers and epicatechin. Following the work of Clifford et al. [ 34 ], the isomeric form of each caffeoylquinic, feruloylquinic and dicaffeoylquinic acids was assigned. Trigonelline and caffeine have been previously identified in aqueous arabica and robusta coffee extracts [ 35 , 36 ]. A higher level of trigonelline as compared to caffeine was found, in agreement with published results [ 37 ]. Trigonelline biosynthesis occurs in the pericarp of coffee berries, whereas caffeine is predominantly produced in the beans [ 37 ]. Interestingly, the NAD + precursor trigonelline seems to improve muscle function during Foods 2025,14, 44 8 of 18 ageing [ 38 ]. Comparable to our findings, protocatechuic and chlorogenic acids were the dominant phenolic compounds identified in a beverage prepared with dried coffee cherry pulp and hot water [ 33 ]. Several caffeoylquinic, feruloylquinic, and dicaffeoylquinic acid isomers have been found by others, too [ 3 , 34 , 36 ]. However, caffeoylferuloylquinic or p-coumaroylquinic acids [3,33] were not detected in this work. Foods 2025, 14, x FOR PEER REVIEW 8 of 19 [32]. The TPC of the CPE was lower than the 1.3 mg GAE / mL obtained for the raw filtrate of wet coffee processing [31]. However, TPC on a weight basis (4.06 mg GAE/g fresh pulp) was in agreement with the 4.9 mg GAE/g pulp DM [33]. Compared to the mangosteen extracts of our previous work [22], CPE contained a much lower TPC (5.70 and 10.01 mg GAE / mL for pulp and rind, respectively). Accordingly, the CPE antioxidant capacity was lower than those of the mangosteen extracts (100.95 and 186.60 mM TE for pulp and rind, respectively). The most abundant phenolic compounds detected in the CPE by HPLC-PAD/HPLCESI-MS (Figure 1) were, in decreasing amounts (Table 4), the alkaloids trigonelline and caffeine, protocatechuic and chlorogenic acids, caffeoylquinic and feruloylquinic acids, the flavan-3-ol catechin and, in lower quantities, caffeic acid, the flavonoid glycoside rutin, several dicaffeoylquinic acid isomers and epicatechin. Following the work of Clifford et al. [34], the isomeric form of each caffeoylquinic, feruloylquinic and dicaffeoylquinic acids was assigned. Trigonelline and caffeine have been previously identified in aqueous arabica and robusta coffee extracts [35,36]. A higher level of trigonelline as compared to caffeine was found, in agreement with published results [37]. Trigonelline biosynthesis occurs in the pericarp of coffee berries, whereas caffeine is predominantly produced in the beans [37]. Interestingly, the NAD+ precursor trigonelline seems to improve muscle function during ageing [38]. Comparable to our findings, protocatechuic and chlorogenic acids were the dominant phenolic compounds identified in a beverage prepared with dried coffee cherry pulp and hot water [33]. Several caffeoylquinic, feruloylquinic, and dicaffeoylquinic acid isomers have been found by others, too [3,34,36]. However, caffeoylferuloylquinic or p-coumaroylquinic acids [3,33] were not detected in this work. (A) 7 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 -ESI EIC(515.1195) Scan Frag=120.0V EC1-4 16-04-24 2ul NEGmsms.d 1 7 Counts vs. Acquisition Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Foods 2025, 14, x FOR PEER REVIEW 9 of 19 (B) Figure 1. Extracted ion chromatogram of major polyphenol compounds detected in the coffee cherry pulp extract. (A): compounds detected after operation in the positive ion mode. (B): compounds detected after operation in the negative ion mode. Table 4. LC-MS/MS characteristics of major polyphenols detected in the coffee cherry pulp extract. Peak Number Retention Time (min) Compound [M-H]- m/z MS2 Ions m/z 1 1.9 Trigonelline 138.0550 * 138 (100), 94 (63), 53 (27), 78 (18), 65 (13) 2 4.1 3-Caffeoylquinic acid 353.0878 191 (100), 135 (35) 3 4.4 Protocatechuic acid 153.0193 109 (100) 4 6.1 Chlorogenic acid (5-Caffeoylquinic acid) 353.0878 191 (100) 5 6.3 Catechin 289.0718 203 (100), 245 (87), 109 (65), 221 (64), 164 (57), 123 (45), 227 (38), 97 (37), 150 (36), 80 (32), 186 (32), 138 (31), 211 (25), 175 (23) 6 6.6 4-Caffeoylquinic acid 353.0878 191 (100), 173 (100), 135 (34) 7 6.7 Caffeine (1,3,7-Trimethylxanthine) 195.0877 * 138 (100) , 195 (41), 110 (19) 8 6.7 3-Feruloylquinic acid 367.1035 193 (100), 134 (19) 9 8.1 Caffeic acid 179.0350 135 (100), 105 (12), 93 (12) 10 8.1 5-Feruloylquinic acid 367.1035 367 (100) 11 8.9 Epicatechin 289.0718 203 (100), 245 (87), 109 (65), 221 (64), 123 (45), 227 (38), 97 (37), 150 (35), 186 (32), 80 (32), 289 (31), 138 (31), 211 (25) 12 10.5 4-Feruloylquinic acid 367.1035 191 (100), 173 (24) 13 14.1 Rutin (Quercetin-3-O-rutinoside) 609.1461 609 (100), 300 (11) 14 14.5 609.1461 609 (100), 300 (17) 15 17.7 3,4-di-O-caffeoylquinic acid 515.1195 173 (100), 353 (83), 515 (37), 335 (19), 155 (13) 16 18.5 3,5-di-O-caffeoylquinic acid 515.1195 353 (100), 191 (71), 179 (14), 509 (10) 17 21.1 4,5-di-O-caffeoylquinic acid 515.1195 353 (100), 173 (69), 311 (14), 516 (10), 263 (10) * Major polyphenol compounds detected in the positive ion mode [M+H]+. Flavonoids such as catechin, epicatechin and rutin are found in many plants. These three compounds were also detected in mangosteen extracts [22]. In agreement with our 6 x10 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45 1.5 1.55 1.6 1.65 1.7 1.75 -ESI EIC(515.1195) Scan Frag=120.0V EC1-4 16-04-24 2ul NEGmsms.d 2 3 4 5 6 8 9 10 12 11 1314 15 16 17 Counts vs. Acquisition Time (min) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Figure 1. Extracted ion chromatogram of major polyphenol compounds detected in the coffee cherry pulp extract. (A): compounds detected after operation in the positive ion mode. (B): compounds detected after operation in the negative ion mode. Flavonoids such as catechin, epicatechin and rutin are found in many plants. These three compounds were also detected in mangosteen extracts [ 22 ]. In agreement with our results, all three were reported in an 80% methanol fresh coffee pulp extract [ 39 ]. However, only rutin was detected in coffee cherry pulp samples [ 30 ]. According to Aravind et al. [ 11 ], these compounds have been shown to improve the function of endothelial tissues, alter glucose metabolism, reduce oxidative stress and ameliorate insulin resistance. Foods 2025,14, 44 9 of 18 Table 4. LC-MS/MS characteristics of major polyphenols detected in the coffee cherry pulp extract. Peak Number Retention Time (min) Compound [M-H]- m/z MS2 Ions m/z 1 1.9 Trigonelline 138.0550 * 138 (100), 94 (63), 53 (27), 78 (18), 65 (13) 2 4.1 3-Caffeoylquinic acid 353.0878 191 (100), 135 (35) 3 4.4 Protocatechuic acid 153.0193 109 (100) 4 6.1 Chlorogenic acid (5-Caffeoylquinic acid) 353.0878 191 (100) 5 6.3 Catechin 289.0718 203 (100), 245 (87), 109 (65), 221 (64), 164 (57), 123 (45), 227 (38), 97 (37), 150 (36), 80 (32), 186 (32), 138 (31), 211 (25), 175 (23) 6 6.6 4-Caffeoylquinic acid 353.0878 191 (100), 173 (100), 135 (34) 7 6.7 Caffeine (1,3,7-Trimethylxanthine) 195.0877 * 138 (100), 195 (41), 110 (19) 8 6.7 3-Feruloylquinic acid 367.1035 193 (100), 134 (19) 9 8.1 Caffeic acid 179.0350 135 (100), 105 (12), 93 (12) 10 8.1 5-Feruloylquinic acid 367.1035 367 (100) 11 8.9 Epicatechin 289.0718 203 (100), 245 (87), 109 (65), 221 (64), 123 (45), 227 (38), 97 (37), 150 (35), 186 (32), 80 (32), 289 (31), 138 (31), 211 (25) 12 10.5 4-Feruloylquinic acid 367.1035 191 (100), 173 (24) 13 14.1 Rutin (Quercetin-3-O-rutinoside) 609.1461 609 (100), 300 (11) 14 14.5 609.1461 609 (100), 300 (17) 15 17.7 3,4-di-O-caffeoylquinic acid 515.1195 173 (100), 353 (83), 515 (37), 335 (19), 155 (13) 16 18.5 3,5-di-O-caffeoylquinic acid 515.1195 353 (100), 191 (71), 179 (14), 509 (10) 17 21.1 4,5-di-O-caffeoylquinic acid 515.1195 353 (100), 173 (69), 311 (14), 516 (10), 263 (10) * Major polyphenol compounds detected in the positive ion mode [M+H]+. 3.3. CPE Effect on Bacterial Growth and Metabolism After 24 h, all tested lactobacilli strains grew just as well in control as in CPEsupplemented BHIC media (Table S1). Strains INIA P495, P708 and P334 obtained the highest values, with levels close to 9 log CFU/mL. Although a stimulatory effect of polyphenols has been described for lactobacilli [ 40 ], no significant differences between both growth media, either at day 1 or 6, were recorded. However, pH decreases were significantly bigger (p< 0.01) in CPE-supplemented BHIC than in BHIC controls. The highest decreases were recorded for strains INIA P495, INIA P708 and GG, with values around 4.3–4.5, in agreement with observations of solid media (Table 2). Although pH values of 4.5–5.0 typically result in a significant to complete inhibition of growth [ 41 ], CPE seemed to protect cultures. We found no differences in growth between CPE-supplemented or control media. Similarly, Pereira et al. [ 42 ] showed that inoculation with a Lp. plantarum strain resulted in faster and improved coffee bean fermentation, converting pulp sugars into organic acids and reaching pH values of 4.5 in 12 h. CPE addition significantly (p< 0.01) increased TPC, by about 100 µ g GAE / mL. However, no increase in TPC values were recorded for any of the tested cultures. While lactobacilli are able to transform complex dietary polyphenols, increasing the bioavailability of these compounds [ 16 , 43 ], the spectroscopic technique used to determine TPC did not seem to be sensitive enough to detect the differences observed by HPLC-ESI/MS [44]. 3.4. Functional and Probiotic Capacities of Selected Strains in the Presence of CPE For the next experiments, three of the six lactobacilli strains, each showing different survival and metabolic behavior, were selected (INIA P495, INIA P708 and INIA P459). 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