Bioactive Compounds, Ruminal Fermentation, and Anthelmintic Activity of Specialty Coffee and Spent Coffee Grounds In Vitro

Abstract

We quantified the bioactive compounds of Ethiopian coffee (ETH), spent coffee grounds SCGs from ETH (SCG-ETH), and mixed SCGs (SCG-MIX) prepared by filtration methods and investigated the effect of SCG-ETH on ruminal fermentation as well as the anthelmintic activity of ETH. Three substrates, meadow hay (MH)-barley grain (MH-BG), MH-SCG-ETH, and BG-SCG-ETH (1:1 w/w), were fermented using an in vitro gas production technique. The bioactive compounds were quantitatively analyzed using ultra-high-resolution mass spectrometry. We performed an in vitro larval development test to determine the anthelmintic effect of an aqueous extract of ETH against the gastrointestinal nematode (GIN) Haemonchus contortus. The total content of bioactive compounds was highest in SCG-ETH, followed by SCG-MIX and ETH (35.2, 31.2, and 20.9 mg/g dry matter, respectively). Total gas and methane production (p < 0.001) were decreased by both MH-SCG-ETH and BG-SCG-ETH. The in vitro digestibility of dry matter was higher for MH-SCG-ETH and BG-SCG-ETH than for MH-BG. The aqueous ETH extract exhibited a strong larvicidal effect, with a mean lethal dose of 13.2 mg/mL for 50% mortality and 31.9 mg/L for 99% mortality. SCG substrates have the potential to modulate ruminal fermentation and serve as a source of anthelmintic bioactive compounds against GINs in ruminants.

1. Introduction

Coffee is one of the most commonly consumed beverages in the world, and its production and consumption have increased over the past decades, particularly with the arrival of the specialty coffee market [1]. Specialty coffee is made from the highest quality green coffee beans, has a known geographical origin, and is produced using the best postharvest processing methods (e.g., natural, washed, honey, fermentation, and carbonic maceration) [2]. It also involves the optimal conditions for storing green beans and is crafted from beans harvested in the best year [3]. Unlike commercial (conventional) coffees, specialty coffee follows a standardized production characterized by the quality and uniqueness of its origin, which involves criteria used for selecting plantations to the brewing methods [4]. Specialty coffees also contain higher total contents of polyphenols compared to conventional coffees [5]. The average loss of total polyphenol content in dark-roasted conventional coffee is nearly 93% [6], but the average losses in specialty coffees are considerably lower [4,7]. The content of bioactive compounds in coffee depends mainly on the origin and processing of the beans, the roasting conditions, and the brewing technique [7,8,9]. Spent coffee grounds (SCGs) are a major by-product of the preparation of coffee beverages produced through various brewing methods such as hot water extraction (e.g., Hario V60, Aeropress, drip brewing, and French press) or steam (espresso). Most of the SCGs are either incinerated or disposed of in landfills, potentially posing a risk to the environment [10]. Coffee processing generates approximately 0.65 kg of SCGs from 1 kg of green coffee and about 2 kg of wet SCGs from 1 kg of instant coffee [11]. However, SCGs can be recovered and reused, enhancing the sustainability of the circular economy by improving waste management in the coffee industry [12]. While the price of SCGs from specialty coffees may differ from that of conventional coffees, SCGs are generally inexpensive, e.g., 0.061 USD/kg for Colombian coffees [13]. SCGs are rich in nutrients and various bioactive components, including good sources of fiber, protein, polyphenols, and lipids, mainly polyunsaturated fatty acids [14,15].

An increasing number of studies have recently attempted to evaluate SCGs as a feed supplement to improve the productive performance of ruminants [16,17]. Some evidence also suggests that SCGs have anthelmintic activity against Haemonchus contortus [18], a parasitic gastrointestinal nematode (GIN) of small ruminants, which may affect several factors associated with ruminal fermentation and methane emissions [19]. The potential of SCGs from specialty coffees, prepared by filtration methods and rich in bioactive compounds, as a feed replacement or supplement in ruminants, however, has not been sufficiently investigated. Given the high content of bioactive compounds in filtered specialty coffees, we hypothesized that SCGs as a feed replacement would strongly affect ruminal fermentation and mitigate methane emissions, and that aqueous extracts of specialty coffee would exhibit anthelmintic activity in vitro. We used Ethiopian specialty coffee (ETH), SCG-ETH, and SCGs from blended specialty coffee (SCG-MIX) prepared by filtration methods. Our objectives were to quantify the bioactive compounds in ETH, SCG-ETH, and SCG-MIX, determine the effect of SCG-ETH on ruminal fermentation characteristics in vitro, and evaluate the anthelmintic effect of the aqueous extract of ETH.

2. Materials and Methods

2.1. Ethics Statement

This study was conducted following the guidelines of the Declaration of Helsinki and national legislation in the Slovak Republic (G.R. 377/2012; Law 39/2007) for the care and use of research animals. The Ethical Committee of the Institute of Parasitology of the Slovak Academy of Sciences approved the experimental protocol on 20 July 2024 (protocol code 2024/27).

2.2. Twenty-Four-Hour In Vitro Batch-Culture Experiment

2.2.1. Experimental Design

The in vitro gas production technique (IVGPT) has been used to simulate ruminal fermentation of feed substrates. Three replicates (three serum bottles in each incubation) were prepared for meadow hay (MH) and barley grain (BG): MH-BG, MH-SCG-ETH, and BG-SCG-ETH. The experiment consisted of fermentations of the three substrates with ruminal content inocula, repeated three times, over three consecutive days (n = 3 × 3). Three replicate bottles were also used for the blank control (inoculum but no substrate).

2.2.2. Inoculum and Substrates

An inoculum of ruminal content (RC, both solid and liquid) was obtained from six Tsigai sheep (10 months old and average weight of 32.7 ± 3.45 kg), which were fed 300 g/kg dry matter (DM) concentrate (MIKROP ČOJ, Čebín, Czech Republic) and 700 g/kg DM MH per day in two equal meals with free access to water. Two sheep were slaughtered per day in a slaughterhouse following the rules of the European Commission (Council Regulation 1099/2009) for slaughtering practices [20], for three consecutive days. The lambs were euthanized by using an overdose of 140 mg/kg of pentobarbital (Dolethal, Vetoquinol, UK, Ltd., abattoir of the Centre of Biosciences of Slovak Academy of Sciences, Institute of Animal Physiology, Košice, Slovakia, No. SK U 06018). Pentobarbital overdose had a negligible effect on the estimated ruminal in vitro parameters. The carcasses were sent to the Department of Pathological Anatomy and Pathological Physiology, University of Veterinary Medicine and Pharmacy in Košice in the Slovak Republic. RCs were immediately transported to the laboratory in a water bath maintained at 39 °C. RCs were passed through four layers of gauze, pooled and mixed 1:2 with McDougall’s buffer [21]. This inoculum (35 mL) was dispensed into each fermentation bottle (100 mL) containing 0.25 g of a substrate. The fermentation bottles were filled with CO₂, closed with a butyl rubber stopper, and sealed with aluminum crimp caps. Then the bottles were incubated in an incubator (Galaxy 170R, Eppendorf North America Inc., Hauppauge, NY, USA) for 24 h at a temperature of 39 °C in an anaerobic condition with periodical mixing of the contents. The experiment was carried out using IVGPT, where the volume of accumulated gas released from the recorded pressure or the volume of gas produced after 24 h of fermentation using a mechanical manometer fitted to a transducer (Premagas, StaráTurá, Slovak Republic) was determined [22]. SCGs from specialty coffees (Coffea arabica) prepared by filtration methods were obtained from a coffee shop (Bølge, Košice, Slovakia) that exclusively sells and prepares specialty coffees using various brewing techniques. SCGs from roasted Ethiopian coffee (SCG-ETH, Ethiopia Konga Natural, Yirgacheffe, Gedeo) and other specialty coffees (SCG-MIX) were analyzed in the study. The SCGs were transported to the laboratory and dried to a constant weight at 40 °C for 24 h. BG, MH, and SCG-ETH were used as substrates for the in vitro experiment (MH-BG, MH-SCG-ETH, and BG-SCG-ETH at 1:1 w/w), where SCG replaced forage or concentrate of the diets. MH, BG, and SCG-ETH were ground through a 0.15–0.40 mm screen using a Molina grinder (MIPAM BIO s.r.o., České Budějovice, Czech Republic) or a stand mixer (Bosch, Berlin, Germany). The experiment took place in the autumn of 2024.

2.2.3. Measurements

The in vitro dry matter digestibility (IVDMD) was determined from the difference in substrate weights before and after incubation. The contents of the batch cultures were transferred to centrifuge tubes and centrifuged at 3500× g for 10 min. The pellets were washed twice with 50 mL of distilled water, centrifuged, and dried to a constant weight at 105 °C for 3 days [23]. Gas samples (1 mL) were collected from the headspace of the bottles using a gastight syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) for measuring the production of methane in the batch cultures. Short-chain fatty acids (SCFAs) and in vitro methane production were analyzed on a Perkin Elmer Clarus 500 gas chromatograph (Perkin Elmer, Shelton, CT, USA) [24]. The pH of the batch cultures was measured using an InoLab pH Level 1 pH meter (Xylem Analytics Germany Sales GmbH & Co., KG, Weilheim, Germany). The concentration of ammonia N (nitrogen) in the inocula was determined using the phenol-hypochlorite method [25]. Samples for counting ciliate protozoa were fixed with an equal volume of an 8% solution of formaldehyde [26].

2.3. Chemical Analyses

2.3.1. Nutritional Analysis

The substrates (i.e., BG, MH, ETH, SCG-ETH, and SCG-MIX) were analyzed in triplicate by standard procedure [27]. The DM content was obtained by drying the samples at 105 °C for at least 24 h in an oven (method no. 930.15). The total ash content of the samples was determined by ashing overnight at 550 °C (method no. 942.05) in a muffle furnace. Nitrogen (N) content (method no. 968.06) was determined using a FLASH 4000 N/Protein Analyzer (Thermo Fisher Scientific, Cambridge, UK). Crude-protein content was calculated by multiplying the total N content by 6.25 (method no. 990.03). The acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents were analyzed as described previously [28] using an ANKOM 2000 Automated Fiber Analyzer (Ankom Technology, Macedon, NY, USA) with heat-stable α-amylase. The chemical compositions of the substrates are presented in

Table 1. Chemical compositions of the dietary substrates (n = 3).

Substrate	DM 1	NDF 2	ADF 3	CP 4	N 5	Ash
	(g/kg)	(g/kg DM)
BG 6	895	169	75	118	19.1	25.6
MH 7	900	452	287	124	19.9	96.2
ETH 8	962	463	291	114	18.2	42.5
SCG-ETH 9	878	602	374	105	16.8	8.21
SCG-MIX 10	908	636	405	109	17.5	11.3

¹ Dry Matter; ² Neutral Detergent Fiber; ³ Acid Detergent Fiber; ⁴ Crude Protein; ⁵ Nitrogen; ⁶ Barley Grain; ⁷ Meadow Hay; ⁸ Ethiopian Coffee; ⁹ Spent Coffee Ground from Ethiopian coffee; ¹⁰ Spent Coffee Ground from Mixed Specialty Coffees.

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2.3.2. Analysis of Polyphenols

All substrate samples (ETH, SCG-ETH, and SCG-MIX) were ground into a fine powder using an FW177 Herbal Medicine Disintegrator at 24,000 rpm (Hangzhou Chincan Trading Co., Ltd., Hangzhou, Zhejiang, China), and 100 mg of each powder were extracted three times with 80% methanol in an ultrasonic bath at 40 °C for 35 min. The particular extracts were combined, evaporated to dryness, dissolved in 2 mL of Milli-Q water (acidified with 0.2% formic acid), and then purified by solid-phase extraction (SPE) using a 60 mg Oasis HLB 3cc Vac Cartridge (Waters Corp., Milford, CT, USA). The cartridges were washed with 0.5% methanol to remove carbohydrates and then washed with 80% methanol to elute the polyphenolic fraction. The polyphenolic fraction was re-evaporated to dryness and dissolved in 1 mL of 80% methanol (acidified with 0.2% formic acid). The sample was then centrifuged (23,000× g for 5 min) and diluted five-fold with Milli-Q water before spectrometric analysis. All extracts were performed in triplicate for two independent samples and stored at −20 °C before analysis. All procedures were repeated three times.

2.3.3. Ultra-High-Resolution Mass Spectrometry (UHRMS)

The compounds from the 80% methanol SPE fraction containing polyphenols were analyzed using UHRMS on a Dionex UltiMate 3000RS system (Thermo Scientific, Darmstadt, Germany) with a charged aerosol detector interfaced with a Compact high-resolution quadrupole time-of-flight mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). The compounds were chromatographically separated using a 100 mm × 2.1 i.d.; 2.6 µm Kinetex C18 column (Phenomenex, Torrance, CA, USA), with mobile phase A consisting of 0.1% (v/v) formic acid in water and mobile phase B consisting of 0.1% (v/v) formic acid in acetonitrile. The initial mobile phase was 7% B and 93% A held for 1 min, then from 1 min to 20 min was ramped concavely from 7 to 50% phase B in phase A to separate the phenolic compounds, with a short 0.3 min calibration segment from 0 to 0.5 min. The flow rate was 0.3 mL/min, and the column was held at 25 °C. Spectra were acquired in negative-ion mode over a mass range from m/z 100 to 1500 with a 5 Hz frequency. The operating parameters of the electrospray ion source were as follows: capillary voltage, 3 kV; dry-gas flow, 6 L/min; dry-gas temperature, 200 °C; nebulizer pressure, 0.7 bar; collision radio frequency, 600.0 V; transfer time, 70.0 µs; and pre-pulse storage, 7.0 µs. Ultrapure nitrogen was used as the drying and nebulizer gas, and argon was used as the collision gas. The collision energy was set automatically from 15 to 75 eV, depending on the m/z of the fragmented ion. The data were calibrated internally with sodium formate introduced to the ion source at the beginning of each separation via a 20 µL loop. The spectra data were acquired and processed using Bruker DataAnalysis 4.3 software (Bruker Daltonik GmbH, Bremen, Germany). The concentrations of the phenolic compounds were calculated as equivalents of chlorogenic acid (CGA). Calibration curves were constructed based on five concentration points (from 0.45 to 0.0021 mg/mL) using Bruker QuantAnalysis 4.3 software (Bruker Daltonik GmbH, Bremen, Germany). All analyses were performed in triplicate.

2.4. Anthelmintic Activity of Coffee Extract

2.4.1. Aqueous Coffee Extract

For the Hario V60 method of extraction, a Hario filter paper (Hario, Koga-Ibaraki, Japan) was placed in a ceramic dripper and rinsed with hot water. Freshly ground beans (15 g) were added to the dripper, and water at a temperature of 94 °C was poured over them. An initial infusion of 40 mL of distilled water (blooming) was used to start the release of CO₂ from the ground beans. An additional 185 mL of water was gradually added after 30 s. The total time was 2 min 30 s. The concentration of the stock solution for the in vitro larvicidal test was 66.7 mg/mL.

2.4.2. Larval Development Test (LDT)

A susceptible isolate of H. contortus was obtained from donor sheep experimentally infected with 5000 infective larvae (L3). The isolate was an inbred isolate of MHCo3 [29]. Nematode eggs were collected by sequential sieving through three stacked sieves with apertures of 250, 100, and 25 µm, as previously described [30]. The test was performed in 96-well microtiter plates in 12 concentrations. The stock solution of aqueous coffee extract was serially diluted 1:2 with distilled water to give 12 concentrations, and aliquots (120 µL) of each concentration or water (controls) were transferred into wells of a 96-well flat-bottomed plate. To each well was added: 10 µL of an egg suspension (approximately 70–100 eggs in the distilled water) containing Amphotericin B (Sigma-Aldrich, Darmstadt, Germany), 20 µL of a culture medium [31], and 120 µL from a stock solution of aqueous coffee extract. The final aqueous concentrations in the test ranged from 53.3 to 0.026 mg/mL. A control (distilled water) was also included in the test. The test was performed with seven replicates for each concentration and control. The plates were incubated at 26 °C for seven days, and the incubation was then terminated by adding 10 µL of Lugol’s solution to each well. The proportions of unhatched eggs and L1–L3 larvae in each well after incubation were counted under an inverted microscope. Larvicidal activity was expressed as the concentration (mg/mL) of mean lethal doses, LD50 or LD99 (concentrations of an aqueous coffee extract that prevents 50 or 99% of larvae from developing to the infective L3 stage, respectively). A logit model of regression analysis was applied to determine the LD50 and LD99 concentrations of the extracts in the LDT.

2.5. Statistical Analysis

Data were analyzed using GraphPad Prism 9.2.0 (332) 2021 (GraphPad Software, Inc., San Diego, CA, USA) using one-way analyses of variance. Differences were determined using Tukey’s multiple-comparison post hoc tests when the overall effect was significant (p < 0.05). The LDT data were analyzed using a statistical logistic regression model to determine LD50 and LD99 [32].

3. Results

3.1. Bioactive Compounds

The peak chromatogram numbers in Figure 1 represent the main bioactive compounds of the ETH, SCG-ETH, and SCG-MIX samples, as numbered in

Table 2. Bioactive compounds (mg/g) in the ETH, SCG-ETH, and SCG-MIX (n = 3, mean ± SD).

No.	RT	UV	m/z [M-H]	Formula	MS2 Main ion	MS2 Fragments	Compound	ETH	SCG-ETH	SCG-MIX	p
1	1,80	215,325	353.0884	C16H18O9	191.0566	179,135,161	cis 3-O-CQA 1	0.47 ± 0.01 a	1.69 ± 0.11 c	0.87 ± 0.01 b	<0.001
2	1,91	277,325	353.0875	C16H18O9	191.0566	179,135,161	trans 3-O-CQA 2	1.71 ± 0.31 a	2.66 ± 0.21 b	2.45 ± 0.41 ab	0.025
3	3,23	215,325	353.0878	C16H18O9	179.0548	173,191,135,161	trans 4-O-CQA 3	0.01 ± 0.00 a	0.20 ± 0.01 b	0.26 ± 0.01 c	<0.001
4	3,80	215,325	353.0887	C16H18O9	191.0558	179,173,135	cis 5-O-CQA 4	3.60 ± 0.71 a	7.35 ± 1.02 b	5.44 ± 0.52 ab	0.003
5	4,27	215,325	353.0884	C16H18O9	191.0564	173,179,135	trans 5-O-CQA 5	9.04 ± 1.05	13.1 ± 4.01	10.5 ± 3.01	0.306
6	5,89	215,325	335.0766	C16H16O9	161.0244	179	cis 4-O-FQA 6	0.11 ± 0.01	0.09 ± 0.01	0.10 ± 0.00	0.064
7	6,26	215,325	353.0874	C16H18O9	191.0563	179,173	cis 4-O-CQA 7	0.00 ± 0.00 a	0.08 ± 0.01 c	0.03 ± 0.00 b	<0.001
8	6,63	215,325	337.0923	C16H18O8	173.0457	163	5-O-pCoCQA 8	0.00 ± 0.00 a	0.04 ± 0.01 b	0.06 ± 0.00 c	<0.001
9	6,83	215,325	337.0924	C16H18O8	191.0545	173,163	4-O-pCoCQA 9	0.04 ± 0.00	0.04 ± 0.01	0.04 ± 0.00	0.422
10	7,06	215,325	337.0924	C16H18O8	191.0545	173,163	3-O-pCoCQA 10	0.02 ± 0.00	0.02 ± 0.01	0.02 ± 0.00	0.422
11	7,18	215,325	335.0768	C16H16O9	161.0245	191	3-LCQA 11	0.04 ± 0.01 a	0.08 ± 0.01 b	0.06 ± 0.01 ab	0.008
12	7,60	215,323	367.1037	C17H20O9	173.0458	193	5-O-FQA 12	0.05 ± 0.00 a	0.20 ± 0.02 b	0.35 ± 0.02 c	<0.001
13	7,78	215,323	367.1041	C17H20O9	191.0567	173,161,134	4-O-FQA 13	0.06 ± 0.00 a	0.26 ± 0.21 ab	0.45 ± 0.01 b	0.022
14	8,10	215,325	335.0778	C16H16O9	161.0251	179	ep-3-LCQA 14	0.85 ± 0.11 a	2.33 ± 0.32 b	2.62 ± 0.71 b	0.007
15	8,19	215,325	335.0774	C16H16O9	161.024	179	3-LCQA 15	2.13 ± 0.23	1.68 ± 0.41	1.64 ± 0.02	0.124
16	8,51	215,325	335.0784	C16H16O9	161.02	179	ep-4-LCQA 16	0.76 ± 0.01 b	0.41 ± 0.03 a	0.45 ± 0.02 a	<0.001
17	8,58	215,325	335.0771	C16H16O9	161.019	179	4-LCQA 17	0.34 ± 0.01 a	0.81 ± 0.02 b	0.88 ± 0.04 c	<0.001
18	9,30	215,325	515.1192	C25H24O12	341.0655	191	3,4-diCQA 18	0.01 ± 0.00 a	0.01 ± 0.01 a	0.06 ± 0.00 b	<0.001
19	10,67	215,325	349.0929	C17H18O8	175.0403	160	4-LFQA 19	0.02 ± 0.01 a	0.05 ± 0.00 b	0.19 ± 0.00 c	<0.001
20	10,96	215,325	515.1199	C25H24O12	353.0869	173,179,191,161,135	3,4-DiCQA 20	0.41 ± 0.01 a	1.10 ± 0.03 b	1.20 ± 0.47 b	0.023
21	11,30	215,325	515.12	C25H24O12	353.087	173,179,191,161,135	4,5-DiCQA 21	0.36 ± 0.03 a	0.85 ± 0.04 b	0.79 ± 0.00 b	<0.001
22	12,06	215,325	515.1201	C25H24O12	353.0869	191,179,173,161,135	3,5-DiCQA 22	0.62 ± 0.02 a	1.43 ± 0.08 b	1.47 ± 0.04 b	<0.001
23	12,32	215,325	529.1339	C26H26O13	193.0591	173,335,161,135	5-C3-FQA 23	0.10 ± 0.01 a	0.15 ± 0.07 ab	0.22 ± 0.03 b	0.043
24	13,10	215,325	529.1346	C26H26O12	193.0587	367,161,134	5-C4-FQA 24	0.01 ± 0.00 a	0.02 ± 0.00 a	0.18 ± 0.02 b	<0.001
25	13,40	215,325	515.1201	C25H24O12	353.0869	191,179,173,161,135	DiCQA 25	0.02 ± 0.00 a	0.07 ± 0.00 b	0.15 ± 0.02 c	<0.001
26	13,70	215,325	529.1345	C26H26O12	173.0434	367,193,155	4-C3FQA 26	0.02 ± 0.00 a	0.05 ± 0.00 b	0.12 ± 0.01 c	<0.001
27	14,9	215,325	497.1065	C25H22O11	335.0768	161	3,5-diLCQA 27	0.05 ± 0.00 a	0.22 ± 0.00 b	0.37 ± 0.01 c	<0.001
28	15,9	215,325	497.1075	C25H22O11	335.0757	179,161,135	5,4-diLCQA 28	0.04 ± 0.00 a	0.17 ± 0.01 b	0.23 ± 0.01 c	<0.001
							Total content:	20.9 ± 0.09 a	35.2 ± 0.24 c	31.2 ± 0.19 b	<0.001

¹ cis 3-O-Caffeoylquinic acid; ² trans 3-O-Caffeoylquinic acid; ³ trans 4-O-Caffeoylquinic acid; ⁴ cis 5-O-Caffeoylquinic acid; ⁵ trans 5-O-Caffeoylquinic acid; ⁶ ep-3-LCQA; ⁷ cis 4-O-Caffeoylquinic acid; ⁸ 5-O-p-Coumaroylquinic acid; ⁹ 4-O-p-Coumaroylquinic acid; ¹⁰ 3-O-p-Coumaroylquinic acid; ¹¹ 3-Caffeoylquinic acid-1,5-lactone; ¹² trans-5-O-Feruloylquinic acid; ¹³ trans-4-O-Feruloylquinic acid; ¹⁴ Epimer 3-caffeoylquinic acid-1,5-lactone; ¹⁵ 3-caffeoylquinic acid-1,5-lactone; ¹⁶ Epimer 4-Caffeoylquinic acid-1,5-lactone; ¹⁷ 4-Caffeoylquinic acid-1,5-lactone; ¹⁸ 3,4-Dicaffeoylquinic acid; ¹⁹ 4-Feruloyl-1,5-quinolactone; ²⁰ 3,4-Dicaffeoylquinic acid; ²¹ 4,5-Dicaffeoylquinic acid; ²² 3,5-Dicaffeoylquinic acid; ²³ 5-Caffeoyl-3-feruloylquinic acid; ²⁴ 5-Caffeoyl-4-feruloylquinic acid; ²⁵ Dicaffeoylquinic acid; ²⁶ 4-Caffeoyl-3-feruloylquinic acid; ²⁷ 3,5-Dicaffeoyl-1,5-quinolactone; ²⁸ 5,4-Dicaffeoyl-1,5-quinolactone. ^a,b,c Within a row, means without a common superscript letter differ at p < 0.05.

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Quantitative analyses of the ETH, SCG-ETH, and SCG-MIX samples identified mainly caffeoylquinic acids (CQAs), coumaroylquinic acids (CoCQAs), feruloylquinic acids (FQAs), dicaffeoylquinic acids (diCQAs), and their isomers (Table 2). In the SCG-ETH substrate, the isomers cis 3-O-CQA, trans-3-O-CQA, and cis-5-O-CQA were present at the highest levels, which were significantly higher (p < 0.001, p = 0.025, and p = 0.003, respectively) than in the ETH and SCG-MIX substrates. The levels of the trans isomer of 5-O-CQA and 3-caffeoylquinic acid-1,5-lactone were also high, but the contents did not differ among the substrates (p > 0.05). The contents of all diCQA isomers were significantly higher in SCG-ETH and SCG-MIX than in ETH.

The contents of many other isomers differed significantly but were relatively low. Most of the CQA derivatives identified in ETH, SCG-ETH, and SCG-MIX were 3,5-diCQA (0.62, 1.43, and 1.47 mg/g DM, respectively), 3,4-diCQA (0.41, 1.10, and 1.20 mg/g DM, respectively), and 4,5-diCQA (0.36, 0.85, and 0.79 mg/g DM, respectively). The total content of bioactive compounds was highest in SCG-ETH, followed by SCG-MIX and ETH (35.2, 31.2, and 20.9 mg/g dry matter, respectively).

3.2. Ruminal Fermentation

The effects of the SCG on ruminal fermentation are presented in

Table 3. Effect of dietary substrates on ruminal fermentation in vitro (n = 9, mean ± SD).

	MH-BG 1	MH-SCG-ETH 2	BG-SCG-ETH 3	p
Total gas (mL/g DM)	289 ± 4.64 b	266 ± 5.47 a	269 ± 4.64 a	<0.001
Methane (mmoL/L)	5.12 ± 0.56 c	2.98 ± 0.66 a	3.80 ± 0.15 b	<0.001
Ammonia N (mg/L)	149 ± 34.6	159 ± 29.2	143 ± 34.3	0.620
IVDMD (g/kg DM) 4	534 ± 1.98 a	545 ± 2.98 b	543 ± 2.37 b	<0.001
pH	6.77 ± 0.07 a	6.68 ± 0.07 a	6.88 ± 0.10 b	<0.001
Total SCFA (mmoL/L) 5	44.6 ± 3.06	47.6 ± 6.19	47.5 ± 2.14	0.233
Acetate (mol%)	60.9 ± 2.03	60.4 ± 2.25	59.6 ± 2.18	0.425
Propionate (mol%)	16.8 ± 0.37 a	18.3 ± 0.81 b	16.8 ± 0.75 a	<0.001
n-Butyrate (mol%)	15.6 ± 1.22 b	13.2 ± 1.32 a	16.1 ± 1.61 b	0.005
iso-Butyrate (mol%)	1.75 ± 0.208 a	2.29 ± 0.180 b	2.19 ± 0.291 b	<0.001
n-Valerate (mol%)	2.73 ± 0.698	2.98 ± 0.842	2.61 ± 1.420	0.742
iso-Valerate (mol%)	2.26 ± 0.380	2.83 ± 0.711	2.80 ± 0.720	0.115
Acetate:propionate	3.63 ± 0.171 b	3.31 ± 0.202 a	3.55 ± 0.181 b	0.003
Total protozoa (103/mL)	108 ± 52.6 b	66 ± 36.2 a	61 ± 39.7 a	0.002

¹ Meadow hay with barley grain (1:1 w/w); ² Meadow hay with spent coffee ground from Ethiopian specialty coffees (1:1 w/w); ³ Barley grain with spent coffee ground from Ethiopian specialty coffees (1:1 w/w); ⁴ In Vitro Dry Matter Digestibility; ⁵ Short-Chain Fatty Acids. ^a,b,c Within a row, means without a common superscript letter differ at p < 0.05.

. The values of total gas (p < 0.001), methane (p < 0.001), IVDMD (p < 0.001), pH (p < 0.001), propionate (p < 0.001), n-butyrate (p = 0.005), iso-butyrate (p < 0.001), acetate:propionate (A:P) ratio (p = 0.003), and total protozoan number (p = 0.002) varied among the substrates tested. For MH-SCG-ETH and BG-SCG-ETH substrates, the values of methane were lower, and values of IVDMD and iso-butyrate were higher compared to the MH-BG substrate. MH-SCG-ETH substrate fermentation produced higher values of propionate and lower values of n-butyrate, and A:P ratio as compared to BG-SCG-ETH and MH-BG substrates.

3.3. LDT

The dose-response relationship of the aqueous filter extracts against H. contortus larval development is shown in Figure 2. The ovicidal and larvicidal effect of the aqueous ETH extracts was already evident after 48 h of incubation. The average egg hatch was about 96% in the control wells, but hatching ranged only between 0 and 20% at extract concentrations of 1.66–6.66 mg/mL. It is noteworthy that although L1 larvae did not hatch after 48 h at concentrations of 1.66–6.66 mg/mL, they were formed, motile, and fully embryonated, and the majority after 7 days reached the L3 stage. On the other hand, ninety to ninety-five percent of the larvae, however, hatched at higher concentrations of 13.3–53.2 mg/mL, but most did not reach the infective L3 stage. LD50 and LD99 were 13.15 and 31.87 mg/mL, respectively.

4. Discussion

There were four main groups of CGAs: CQA, CoCQA, FQA, and diCQA. The contents of the cis-5-O-CQA and trans-5-O-CQA isomers were higher in all samples compared to the other isomers detected, but the content of the cis-5-O-CQA isomer was much higher in SCG-ETH than in ETH. The 5-CQA isomer was generally the most abundant CGA, which forms beneficial bioactive compounds in coffee beans. It is also a major indicator of the quality of beans and contributes to various health-promoting natural products [33]. The CGA content, especially 5-CQA, however, depends on the origin of the beans, post-harvest processes, roasting conditions, and climatic conditions [34,35]. Our results for the content of the 5-CQA isomer in the SCGs, therefore, also differed greatly from those reported in the literature, e.g., 0.83 mg/g [36], 51–201 mg/g [37], and 1.67–3.85 mg/g dry weight [38]. Most of the CQA derivatives identified in ETH, SCG-ETH, and SCG-MIX have biological activity [39].

The total content of bioactive compounds, however, was higher in SCG-ETH than in SCG-MIX and ETH. However, coffee brewing cycles can increase the moisture content and porosity in SCG and induce the release of more of these compounds [40]. Ethiopian coffees are amongst the highest quality coffees [41]. Our results for the bioactive compounds of ETH and SCG-ETH were consistent with other studies comparing SCGs from different geographical regions, including Ethiopia [38,42]. SCG from brewed coffees, especially filtered coffees, as in our study, should generally have a higher content of bioactive compounds than espresso coffees [43,44], although different hydrothermal brewing cycles may affect the bioactive compounds and antioxidant capacity of SCG [40]. However, all three samples (ETH, SCG-ETH, and SCG-MIX) exhibited potential biological activities with beneficial health effects due to the bioactive CGA isomers, and they likely represent a rich source of multitargeted, synergistic therapeutic or prophylactic compounds [45,46].

The ruminal fermentation can be modified by supplementing diets with feed additives containing bioactive compounds [24]. Similarly, SCGs are also rich in bioactive organic compounds (e.g., polyphenols and polysaccharides), which can strongly affect ruminal fermentation [38,47]. Both MH-SCG-ETH and BG-SCG-ETH in our study exhibited high concentrations of anti-methanogenic compounds, including the 5-CQA and 3-CQA isomers [48]. Methane emissions from the ruminal fermentation of MH-SCG-ETH and BG-SCG-ETH, therefore, decreased significantly by 41.8 and 25.8%, respectively, when SCG was used as a replacement for hay or concentrate. This finding is consistent with a previous study in which using SCG as a hay or concentrate replacement significantly reduced methane production by 12.9% [49]. The significant reduction in methane production during fermentation of both SCG substrates in our study was also accompanied by lower total gas production compared to the MH-BG diet, which could be related to the observed reduced methane production. The lower methane production values were consistent with previous results where a diet rich in plant bioactive compounds was fermented in an in vitro experiment [50,51]. The propionate concentration in MH-SCG-ETH, however, increased, probably shifting the ruminal fermentation towards propionic acid production, resulting in less H+ and, therefore, reducing methane production [52]. The fermentation of MH-SCG-ETH was also accompanied by a larger decrease in the total number of ciliated protozoa, n-butyrate values, and A:P ratios compared to the fermentation of MH-BG and BG-SCG-ETH.

Protozoan-associated methanogens contribute approximately 37% to ruminal methane emissions [53] by attaching to protozoan cells as ectosymbionts or by intracellular colonization as endosymbionts [54]. The reduction in the number of protozoa caused by MH-SCG-ETH and BG-SCG-ETH, therefore, might contribute to the reduction in the abundance of methanogens and ultimately methane production. Ciliated protozoa can affect the diversity of ruminal bacteria and fermentation products [55], suggesting that SCGs may serve as promising substrates for modulating ruminal fermentation. An in vivo study found that even a low dose of SCGs (30 g/kg DM) in the concentrate diet of dairy sheep induced a shift in the ruminal bacterial community and an altered correlation between bacterial population and ruminal SCFA concentration [56]. Results in goats indicated that including up to 200 g/kg of SCGs in the concentrate reduced methane emissions per kilogram of organic matter intake due to a reduction in the digestibility of substrates [57]. In this study, a decrease in methane production was accompanied by increased digestibility of SCG substrates, which contrasted with the study [49]. The digestibility of SCG substrates in some studies may be reduced by affecting cell-wall degradation by microbial enzymes due to the Maillard reaction and high-temperature damage during the preparation of coffee beverages [58]. Our experiment did not confirm this possibility, probably because filtered coffee is typically made using water at temperatures of 88–95 °C [59]. Pre-treatment by grinding may also likely improve the digestibility of SCGs and their fermentation in the rumen [17]. Ruminal microbial fermentation of SCGs may also likely improve the digestibility of substrates [15]. Some phenolic compounds are important antinutritional factors [60], but Table 1 indicates that the chemical composition of all dietary substrates had optimal nutritional value and that the SCG substrates did not adversely affect digestibility or ruminal-fluid pH. The overall pH values during the ruminal fermentation of SCG substrates ranged from 6.6 to 6.7 [49,61], which was consistent with our results. Bioactive compounds in SCGs may likely have different effects on ruminal fermentation depending on various factors that can also affect the quality of a cup of coffee, such as origin, environment, cultivation, variety, processing, harvest time, storage, roasting, and brewing [62].

Phytogenic bioactive compounds represent one of the most promising alternatives to synthetic drugs in the treatment of GINs. A strong anthelmintic effect (100% L3 inhibition) of the aqueous extract of ETH was noted in this study. We have previously reported that a broader spectrum of aqueous plant extracts used individually had strong ovicidal and larvicidal activities [63]. Methanolic extracts from plant mixtures, however, have a much stronger anthelmintic effect than aqueous extracts [64]. Aqueous or ethanolic plant extracts with bioactive compounds are generally effective in either suppressing hatching or larval development to the L3 stage [63,65]. The anthelmintic activity of a hydroalcoholic extract of coffee pulp (200 and 100 mg/mL) inhibited hatching of nematodes (100% inhibition), but no larvicidal effect was detected [66], whereas a chloroform extract of C. arabica (200 μg/mL) had effective anthelmintic activity compared to aqueous and ethanolic extracts [67].

Various in vitro tests (EHTs, larval exsheathment inhibition tests, and larval mortality tests) have demonstrated different efficacies of aqueous/acetone extracts of SCGs on the eggs and larvae of H. contortus [18,68]. The effect on development from the L1 to the L3 stage using LDTs, however, has not yet been tested. In addition to tannins and flavonoids, some plants with anthelmintic activity also contain CGAs, especially 1,3-, 3,4-, and 3,5-diCQA isomers [69] or 1,5- and 4,5-diCQA isomers [70]. CGAs obtained from a methanolic extract of Tagetes filifolia completely inhibited the hatching of H. contortus eggs [71]. These results generally clearly indicate that CGA-containing extracts exhibit anthelmintic activity. The anthelmintic activity of SCGs from C. arabica against H. contortus egg hatching and larval development have also indicated inhibition from 53.7 to 100%, and SCGs in the concentrate diet for sheep and goats (10 and 40%, respectively) have decreased the number of eggs in feces from 25 to 69% [18]. These findings indicate that using substrates with bioactive compounds, even those with the best anthelmintic properties in vitro, may not have sufficient effects in vivo. SCGs from specialty coffees, which have a higher total CGA content than does ETH, are likely to have greater anthelmintic effects. In vivo studies are, therefore, needed before SCGs from filtered specialty coffees rich in bioactive compounds are incorporated into the diets of small ruminants with GINs.

5. Conclusions

Our research has highlighted the potential of spent coffee grounds from filter-brewed specialty coffees as a feed substitute for ruminants. Due to their bioactive compounds and relatively low cost, they could be used as alternative sources of feed with the potential to modulate ruminal fermentation, reduce methane emissions, and increase the digestibility of dietary substrates. They are also sources of bioactive compounds with anthelmintic activity, which may be suitable as an alternative for controlling GINs in ruminants. However, in vivo studies are needed to confirm the effect of spent coffee grounds, because bioactive compounds may interact differently with the ruminal microbiota when used long-term as a source of anthelmintic bioactive compounds against GINs in ruminants.