4.1. Nutritional Quality
Considering the nutritional profile of CS, this by-product has a great potential for use as a novel food ingredient [
10,
28,
29,
30]. The results obtained in the present study suggest that the aqueous extract obtained from CS can be considered a ‘source of proteins’ since it contains over 12 g/100 g of proteins as stated in Regulation (EU) No 1924/2006 [
13] (
Table 1). Protein values of CSE are similar to those of egg (12%) and higher than the protein content in milk (3%), as reported by the United States Department of Agriculture (USDA) National Nutrient Database. According to Commission Regulation (EU) No 432/2012, the health claims for foods that are a source of proteins could be applied to CSE. These health claims are as follows: ‘protein contributes to the growth and maintenance of muscle mass’ and ‘to the normal maintenance of normal bones’ [
31]. The extract did not have a significant effect (
p > 0.05) on protein intake biochemical parameters determined in blood samples from rats, urea and creatinine, compared to control rats (
Table 4). The values obtained for these biomarkers were within the physiological range [
32].
To the best of our knowledge, this is the first study that shows the amino acid profile of CSE. The proportion of essential amino acids (34–37%) makes CSE a good source of indispensable amino acids (
Table 1). The claimed effects related to amino acids are growth or maintenance of muscle mass, maintenance of normal muscle function, faster recovery of muscle function/strength/glycogen stores after exercise, faster recovery from muscle fatigue after exercise and skeletal muscle tissue repair [
33]. However, these claimed effects are not established in terms of a cause–effect relationship and have been evaluated by the EFSA Panel with an unfavorable opinion [
33]. CSE may be used as a novel ingredient in foods as a source of amino acids.
As the fat content of CS was 0.44%, this extract could also be considered a ‘low fat’ product [
13]. Fat content in CSE was lower than previously described for CS, which ranged from 1.6% to 3.3% [
3,
34]. Fatty acid composition of CS mainly includes palmitic acid (C16:0), followed by linoleic acid (C18:2n6) and behenic acid (C:22:0), which agrees with that previously reported by other authors [
35]. As expected, the repeated intake of CSE (1 g/kg b.w.) did not have an effect on fat consumption biomarkers, such as cholesterol, aminotransferases and bile acids (
Table 4) in rats treated with CSE compared to the control group.
The total amount of simple carbohydrates composing CSE (6.58 g/100 g) did not rise (
p > 0.05) the glucose levels of treated rats compared to control rats (
Table 4). The amount of total simple sugars is close to the value stated by the European Commission of 5 g of sugars per 100 g for reaching the nutrition claim of ‘low sugar’ [
13]. The value of simple sugars obtained in this study is similar to that obtained by Costa et al. (2018) [
3]. In contrast, this value is higher than that reported for a mixture of Arabica and Robusta CS by Toschi et al. (2014), although the main simple carbohydrates detected in both studies were fructose and sucrose [
35].
The amount of total dietary fiber obtained in this study (22%), as well as SDF (14%) and IDF (8%), agrees with that previously reported for CSE (28%, 24% and 4% for TDF, SDF and IDF, respectively) [
11]. Different authors have reported values of approximately 60%, 50% and 8% of total, insoluble and soluble dietary fiber of CS, respectively [
3,
5,
10,
28]. Ballesteros et al. (2014) reported that insoluble dietary fiber in CS is composed of cellulose, hemicellulose and lignin [
5]. This potential novel ingredient can reach the nutrition claim of ‘high in fiber’ whereby the product must contain at least 6 g of fiber per 100 g [
13]. The health claims attributed to the “high in fiber” nutrition claim are ‘fiber increases fecal bulk, contributes to normal bowel function and to an acceleration of intestinal transit’ [
31].
The WHO recommends a potassium intake of at least 90 mmol/day (3.5 g/day) for adults to reduce blood pressure and the risk of cardiovascular disease, stroke and coronary heart disease [
36]. In this sense, CSE would be a good source of potassium (5.6 g/100 g) [
37], in accordance with Costa et al. (2018) [
3]. According to Commission Regulation (EU) No 432/2012, foods that are a source of potassium can be labeled under the following health claims: “potassium contributes to normal functioning of the nervous system, to normal muscle function and to normal blood pressure” [
31].
On the other hand, the studied extract may also be considered a ‘source of magnesium, calcium and vitamin C’. Recommended daily allowances (RDAs) for magnesium, calcium and vitamin C are 300 mg, 800 mg and 60 mg, respectively [
37]. Since values of magnesium, calcium and vitamin C present in CSE represent 15% of the recommended allowance per 100 g of product, this by-product may be considered a source of these compounds. The nutrition claim source of magnesium is related to the following health claims: “Magnesium contributes to a reduction of tiredness and fatigue, to electrolyte balance, to normal energy-yielding metabolism, to normal functioning of the nervous system, to normal muscle function, to normal protein synthesis, to normal physiological function, to the maintenance of normal bones and teeth and a role in the process of cell division” [
31].
Calcium is also involved in the health claims: “Calcium contributes to normal blood clotting, to normal energy-yielding metabolism, to normal muscle function, to normal neurotransmission, to normal function of digestive enzymes, has a role in the process of cell division and specialization and is needed for the maintenance of normal bones and teeth” [
31].
Finally, health claims regarding vitamin C content are: “Vitamin C contributes to normal function of the immune system, to normal collagen formation for the normal function of blood vessels, bones, cartilages, skin and teeth; contributes to normal energy-yielding metabolism, to normal functioning of the nervous system, to normal physiological function, to the protection of cells from oxidative stress, to the reduction of tiredness and fatigue, to the regeneration of the reduced form of vitamin E and increases iron absorption” [
31].
The strong correlation between diet and health, together with sedentary lifestyles, an aging population and increasing healthcare costs have driven the interest of research in developing healthier food products [
38]. Fiber-enriched foods have been developped in recent years to increase dietary fiber consumption to reduce the risk of chronic diseases. CS has been employed as dietary fiber to reduce caloric density and increase the dietary fiber content of breads [
30]. CS has been added to bread formulations as a natural sustainable source of antioxidants, α-glucosidase inhibitors and colorants [
39]. It has also been used as a coloring and as a dietary fiber source to achieve healthier, nutritious and high sensorial quality biscuits. The nutritional value and appearance of the biscuits also improved by the addition of CS [
40]. CS has also been used in cakes, formulated with up to 30% of water-treated CS as a flour substitute [
12,
41]. In addition, anti-obesity and antioxidant beverages have been developed with CSE. Beverages made from Arabica and Robusta CSE (100 µg/mL) reduced body fat by 21% and 24%, respectively, in
Caenorhabditis elegans as an animal model [
28]. Finally, recent studies have used CS in yogurt production and showed that the bioactive compounds are still bioaccessible after the digestion process [
42].
4.2. Safety
To the best of our knowledge, no study regarding the effects of prolonged exposure to an aqueous extract of CS has been performed. In the present study, CSE from Arabica coffee beans was evaluated in Wistar rats exposed to 1 g/kg b.w. The feeding of CSE (1 g/kg b.w.) to male and female Wistar rats for 4 weeks did not cause mortality in any of the animals. The absence of mortality is considered a positive aspect to support safe use of CSE in animals and in subsequent clinical trials with formulations containing this material.
The safety of these novel foods must be scientifically proven for human consumption. Previous studies have proven the safety CSE at different levels through genotoxicity and acute toxicity studies [
19,
43]. CSE did not induce either cytotoxicity or genotoxicity and protected human cells from DNA strand breaks and oxidative DNA damage effects of chemicals agents such as benzo(a)pyrene [
43]. Furthermore, no lethal effects were observed in acute toxicity studies when rats were treated with 2 g/kg b.w. by oral administration [
19].
With regard to food and water intake, there were no significant differences (
p > 0.05) between treated and control groups, which indicates that CSE does not interfere with these parameters. Weight loss is considered of toxicological importance when the reduction is at least 10% less than the initial body weight [
44]. As expected from the results obtained for food intake, the body weight of rats treated with CSE did not change compared to the control group during the 28 days of the study (
p > 0.05), suggesting that CSE does not compromise nutrient absorption. Other authors have reported the absence of changes in food intake and body weight between control rats and rats treated with β-glucans extracted from barley for 28 days [
45]. El Kabbaoui et al. (2017) also studied the effect of a vegetal aqueous extract on Wistar rats, and no significant changes in body weight were reported when rats were fed with 1 g/kg of
Cistus ladaniferus L. extract [
46].
Significant changes in absolute and relative organ weight of rats can be considered important evidence of toxicity [
47]. The organs in this study were selected according to the Society of Toxicologic Pathology (STP) recommendations. With regard to the macroscopic morphological analysis, there was no change in the shape or weight of the studied organs (thymus, lungs, liver, kidneys, adrenal glands, sex organs, brain, heart and spleen) in rats treated with CSE (1 g/kg b.w.). Furthermore, the histopathology examination showed no histopathological lesions in the liver, kidneys or heart after treatment with CSE. The assessment of histopathological alterations in organs is considered a basic test in the safety assessment of tested materials [
48]. These results reinforce the findings of the absence of toxicity after treatment with 1 g/kg b.w. of this compound for 28 days. Other authors have also reported the absence of morphological or histopathological alterations when Wistar rats were treated with different plant extracts following the same procedure as that described in this study [
46,
49,
50].
The analysis of blood parameters in animal models has a high predictive value for alterations of the hematological system in human toxicity [
51]. No significant differences (
p > 0.05) were observed in biochemical parameters when the diet of rats was supplemented with CSE at 1 g/kg b.w. Glucose, cholesterol, proteins, potassium, sodium, albumin and bile acid levels in treated and control rats were in the same range of that previously described for Wistar rats [
32,
52,
53]. Kidney and liver functionality were evaluated by the measurement of urea and creatinine, and ALT and GGT, respectively. No significant changes (
p > 0.05) were observed in these parameters when animals were treated with CSE 1 g/kg b.w. and the obtained values were similar to those in the reference databases [
32,
52,
53]. No signs of acute of prolonged hepatotoxicity were observed since liver enzyme levels neither increased nor decreased [
54]. These results are in accordance with the absence of toxicity observed in the histopathological analysis of kidney and liver tissue of rats treated with CSE 1 g/kg b.w. for 28 days. On the other hand, damage or destruction of blood cells negatively affects the normal functioning of the body in both humans and animals [
48]. The analysis of erythrocytes, leukocytes and platelets showed the absence of alterations in these parameters after treatment with CSE as well.
4.3. Effect on Key Biological Functions
With regard to hormone secretion, no significant differences (
p > 0.05) were observed in insulin serum levels of healthy rats treated with CSE 1 g/kg for 28 days (
Table 5). However, CSE has shown the ability to modulate insulin secretion in vitro in pancreatic INS-1E cells [
55]. Doses of CSE of 1–10 μg/mL stimulated insulin secretion and reinforced antioxidant defense in pancreatic beta cells stressed with streptozotocin [
15]. Previous studies have also shown the anti-diabetic properties of CSE [
15,
55]. Daily administration of CSE before the induction of diabetes with streptozotocin–nicotinamide (type 2 diabetes model) significantly reduced (
p < 0.05) pancreatic oxidative stress, protecting rats from developing diabetes [
15].
The essential amino acid tryptophan, which is present in CSE (
Table 1), is the precursor of several important products, including serotonin or melatonin [
56]. Serotonin and melatonin are both hormones that regulate various biological functions, such as sleep, appetite and mood. The melatonin-serotonin pathway affects appetite and digestive processes by endocrine as well as paracrine effects in both the brain and the gastrointestinal tract [
57]. The CSE used in this study has 3.4 mg/g dry matter of melatonin [
58]. However, no significant changes (
p > 0.05) were observed in melatonin levels or in its precursor, serotonin, in the serum of rats treated with CSE compared to the control group. CSE may be used as a source of tryptophan, which will act as a neurometabolite.
The caffeine content in CSE is 24 mg/g [
19], and it has been reported that caffeine also impacts circadian rhythms [
59]. In humans, acute caffeine intake has been shown to delay the onset of melatonin secretion and decrease nighttime melatonin levels [
60]. The results obtained in this study indicate that the physiological functions determined by these hormones were not affected by the repeated intake of CSE at 1 g/kg b.w.
Although CSE is known to have a high antioxidant capacity [
10], non-enzymatic and enzymatic oxidative stress biomarkers (GPx, GR, SOD and CAT) analyzed in serum samples were not altered after the administration of CSE 1 g/kg for 28 days (
Table 5). In contrast, previous studies have shown the antioxidant properties of CSE in vivo in pancreatic tissue samples of diabetic rats [
61]. Further studies should be carried out to investigate the antioxidant properties of CSE in certain organs, since polyphenols are metabolized in tissues and these metabolites can also have antioxidant properties [
62]. In addition, no significant differences (
p > 0.05) were observed in inflammation biomarkers, C reactive protein, in treated rats compared to the control group. Recent studies have reported that phenolic compounds composing CSE possess anti-inflammatory properties in vitro. CSE reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and decreased the secretion of pro-inflammatory factors in LPS-stimulated RAW2643.7 macrophages [
63]. Further research regarding the analysis of several pro-inflammatory biomarkers should be carried out in vivo.
Considering the potential fiber effect of CSE, no significant changes (
p > 0.05) in feces number or weight were observed in female rats between groups after 28 days of CSE ingestion (
Table 6). However, male rats treated with CSE showed a lower number of feces compared to the control group. Previous studies have shown that the high molecular weight of CSE, rich in melanoidins, accelerated intestinal transit in treated animals after 28 days of exposure [
64].
CS contains higher amount of soluble dietary fiber compared to other materials, and therefore, can be fermented, and it possesses a large water retention capacity, promotes the growth of bifidobacteria, and decreases the absorption of fat and sugars [
5]. Results regarding the analysis of SCFAs showed that sex seems to influence the SCFAs profile of rats’ feces. Total SCFAs were significantly higher (
p < 0.05) in female control rats compared to male control rats. SCFAs values and molar ratios of acetate:propionate:butyrate (which are also an indicator of dietary changes) of male rats obtained in this study are similar to those previously described [
65]. Shastri et al. (2015) have previously reported the influence of sex in gut fermentation. These authors illustrated the need of considering sex in research studies that investigate health impacts from the intake of functional foods or ingredients that contribute to the improvement of gut health [
66]. Male rats treated with CSE showed a significantly higher (
p < 0.05) content of total SCFAs and acetate than male control rats. This is in agreement with the increased fiber consumption in male rats (
Table 2). The higher levels of SCFAs had no significant effects (
p > 0.05) on the pH values obtained in the feces of rats treated with CSE (
Table 6). Health-promoting properties associated to acetate are increased colonic blood flow and enhanced ileal motility [
67]. Considering the results obtained from the dietary fiber effect and the health claim made on foods high in fiber, it could be said that the dietary fiber present in CSE may contribute to normal bowel function.
With regard to butyrate, there is increasing evidence that this SCFA per se may be beneficial for human health [
68]. Lower values of butyrate (
p < 0.05) were observed in animals treated with CSE. The majority of SCFAs are rapidly absorbed by the colonocytes in the cecum and large intestine, and only approximately 5% are secreted in the feces. The major part of butyrate is used as fuel for colonocytes and it plays an important role in maintaining colonic health in humans [
69,
70]. Therefore, the butyrate produced by fermentation of the fiber present in CSE may have been absorbed by intestinal cells where it exerts potential health benefits. Fiber from another coffee by-product, spent coffee grounds (SCGs), has shown anti-inflammatory properties after in vitro fermentation by human gut microflora. SCFAs produced by colonic fermentation of SCGs exhibited great anti-inflammatory properties by suppressing NO production, and they inhibited inflammatory cytokines [
71]. Considering the results obtained in this study, CSE might be considered a sustainable novel food ingredient with health-promoting properties and beneficial effects on gut microbiota.