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Introduction
Modern animal production is characterized by its great productive intensity, which implies that animals are subjected to different stress situations, which cause imbalance in the intestinal microbiota, the development of pathogenic microorganisms, immunosuppression, as well as the inefficient food conversion, high mortality and decrease in zootechnical response (Huang et al. 2018). For the previous reasons, antibiotics have been used for decades as animal growth promoting additives. However, its indiscriminate use causes residual effects on foods, microbial resistance, damage to the gastrointestinal biota, among others (Eng et al. 2015).
Since 2006, in the European Union and in other countries of the world, the use of sub-therapeutic antibiotics was prohibited (Ronquillo and Hernández 2017). The scientific community and the livestock industry study and introduce new safe and harmless additives to improve animals productivity, such as nutraceutical products (Liu et al. 2016). The American Veterinary Council of Nutraceuticals shows that they are products that contain, in an integral, purified or extracted way, compounds necessary for a correct biological development (Telrandhe et al. 2012).
Among the sources with great potential for obtaining nutraceutical additives, there are plants belonging to the Agave genus. Its chemical composition varies according to edaphoclimatic conditions, soil characteristics, among other factors (García et al. 2010). Generally, it is considered that these plants, due to their high concentration of fructans and other chemical substances, have medicinal properties and model the intestinal microflora and immunity, which causes beneficial effects for the production and health of the host (Adhikari and Kim 2017). Its use in the diet, in small concentrations, could improve the biological and health indicators in animals (Iser et al. 2016a and Valdovinos et al. 2019).
Nowadays, nutraceuticals are one of the most studied products from the physical-chemical point of view, especially to comply with international laws, since they must provide temporary stability, reproducibility, quality, safety and efficacy (Spanish Nutraceutical Society 2015). Previous results reported on the chemical-physical composition of stems meal from A. fourcroydes (SMAF) (Iser et al. 2016b). However, there was not information on the phytochemical compounds and the quality and sensory indicators of this natural product. The objective of this study was to determine the secondary metabolites, quality indicators and organoleptic characteristics of the stems meal from Agave fourcroydes (Henequen) grown in Cuba.
Materials and Methods
Sample preparation. Five henequen stems (A. fourcroydes Lem.) were collected in the early hours of the morning, according to the diagonals method, in the field of the enterprise “Eladio Hernández León”, Matanzas province, Cuba. The average age of these plants was nine years, without inflorescence. This area is characterized by a subtropical climate, Lithosol soil (García-Curbelo et al. 2015) and temperature between 23 and 28 oC (data from Estación Meteorológica “Indio Hatuey”).
The stems of A. fourcroydes had an average weight of 6.39 kg (± 0.32). The outer parts of their bark were stripped with a traditional machete and chopped. The samples were spread on an aluminum tray. Subsequently, they were washed three times with distilled water to remove the greatest amount of impurities.
The drying was carried out first naturally for three days, at room temperature. Then, to obtain a homogeneous drying, they were dried artificially with the help of an oven (WSU 400, Germany), at a temperature of 60°C for 72 h. The samples were milled with a 1 mm diameter sieve, in a hammer mill (Culatte typs MFC), until the final product was obtained, which was stored for six months at room temperature, in fully sealed plastic bags (Mas et al. 2018).
Phytochemical screening. Phytochemical screening was determined according to Payo (2001) methodology. To achieve the highest depletion of the sample, successive extractions with solvents of increasing polarity were performed. A total of 10 g of sample were weighed on an analytical balance (BS 2202S Sartorios, China) and 50 mL of petroleum ether was added. After 48 h, the extract was filtered. To the remainder, 50 mL of 70% ethanol was added to obtain the alcoholic extract and the same was done. 50 mL of distilled water was added and the extraction was performed in an analogous way.
Ultrasound (Ultrasonic Cleaner SB-3200 DTD, China) was applied at 40°C, with a frequency of 40 KHz for two hours, recommended time for optimal extraction (Torres et al. 2014). Secondary metabolites were determined in each extract which, due to their solubility, could be extracted in these solvents.
In the ether extract the tests Mayer (alkaloids), Baljet (coumarins) and Sudan III (fatty acids) were performed. In the ethanolic, Liebermann-Burchard (triterpenes or steroids), foam (saponins), ninhydrin (free amino acids), Mayer (alkaloids), Baljet (coumarins), Fehling (reducing carbohydrates), ferric chloride (phenols and/or tannins), Borntrager (quinones), Shinoda (flavonoids), resins, anthocyanidins were determined, as well as bitter and astringent principles.
In the aqueous extract, the Foam (saponins), Shinoda (flavonoids), Fehling (reducing sugars), Ferric chloride (phenols and/or tannins), Mayer and Wagner (alkaloids), Borntrager (quinones) tests were analyzed. Those of mucilages and bitter and astringent principles were also developed. As a measurement criterion, the crossing system was used to specify the qualification of secondary metabolites.
Quality indicators. In the aqueous extract, the pH was determined in a pH- meter (HANNA 211, Portugal), as described in NC-86-01 (1981). The refractive index was calculated according to WHO Pharm 92.559 (1992) using a refractometer at 200C (ABBE WYA-2S, China). The apparent density was determined in a densimeter at 20°C (TGL 0-12792, Germany) (NC 119: 2001). To specify the acidity, the procedure was according to NC-71: 2000 (2000) and for soluble solids (ABBE WYA-2S, China), in accordance with NC-2173: 2001 (2001).
The phytochemical screening and quality tests of the SMAF were performed in triplicate in the Laboratorio de Productos Naturales belonging to the Centro de Estudio de Química Aplicada, Granma University, Cuba.
Organoleptic characteristics. In addition, in the SMAF was examined the appearance, dissolution capacity, pulverization degree, color, odor, taste and homogeneous capacity, according to the Mendoza and Calvo (2010) methodology.
The organoleptic characteristics of the SMAF were determined in triplicate in the Laboratorio de Química Sanitaria de los Alimentos del Centro Provincial de Higiene, Epidemiología y Microbiología (CPHEM) Granma, Cuba.
Statistical analysis. The data of the SMAF quality indicators were processed using the descriptive statistics module. The mean, standard deviation (SD) and coefficient of variation (CV) were determined. The statistical software SPSS, version 17.0 (2012) was used.
Results and Discussion
The phytochemical screening of SMAF qualified a great variety of secondary metabolites. The ether extract showed coumarins (++) and fatty acids, but not alkaloids (table 1). In both extracts (aqueous and ethanolic), there were not bitter and astringent principles. However, saponins, reducing sugars, alkaloids and flavonoids were detected. The latter had an abundant presence (++) in the aqueous extract. Also the ethanolic extract revealed anthocyanidins (++), coumarins, free amino acids and phenols and/or tannins, without triterpenes or steroids, quinones and resins. In addition, mucilages were identified in the aqueous extract, without phenols or tannins.
Secondary metabolites are natural non-fibrous substances, generated as a defense mechanism against the attack of molds, bacteria, insects and birds, or in some cases, products of plant metabolism under stress conditions (Zandalinas et al. 2017). Phytochemical studies with Agave spp. qualified various beneficial secondary metabolites in leaves, stems and roots. In the Agave americana and Agave barbadensis, new flavonoid molecules were identified (Tinto et al. 2005) and in the Agave tequilana there were flavonoids and phenolic oxhydryls, without the presence of saponins (Flores and Borredon 2013).
Table 1 Phytochemical screening of ether, ethanolic and aqueous extracts of the stems meal from Agave fourcroydes
| Metabolites | Extracts | ||
|---|---|---|---|
| ether | ethanolic | aqueous | |
| Fatty acids | + | ||
| Alkaloids (Mayer) | - | + | + |
| Alkaloids (Wagner) | + | ||
| Coumarins | ++ | + | |
| Saponins | + | + | |
| Flavonoids | ++ | + | |
| Anthocyanidins | ++ | ||
| Triterpenes or esteroids | - | ||
| Mucilage | + | ||
| Reducing carbohydrates | + | + | |
| Quinones | - | ||
| Resins | - | ||
| Free amino acids | + | ||
| Phenols and/or tannins | + | - | |
| Bitter and asrigent principles | - | - | |
Legend: (-) absence; (+) little presence; (++) abundant presence; control no analysis
Several types of steroidal saponins were also found in the Agave fourcroydes, Agave macroacantha and Agave sisalana (Hamissa et al. 2010), while in the Agave intermixa there was a high presence of polyphenolic compounds (García et al. 1999). In other products rich in fructans, such as yacon (Smallanthus sonchifolius) root, mainly reducing carbohydrates and alkaloids were discovered (de Andrade et al. 2017).
In the scientific literature of animal science, secondary metabolites are considered anti-nutritional factors because they exert effects contrary to what is considered an optimal nutrition of animals, especially due to the decrease in digestive metabolism (Savón et al. 2007). However, it has been shown that these secondary metabolites, in small concentrations in diets, improve nutrient digestibility, immune stability, as well as competitive exclusion of microorganisms and intestinal health (Aroche et al. 2018).
Flavonoids (++) detected by phytochemical screening in SMAF (table 1) constitute polyphenolic compounds that, when they are in small concentrations in diets, are beneficial for their antioxidant effect (free radical capture RH *), anti-inflammatory, antiviral and antiallergic, in addition to influencing on the oxidation of LDL and in the regulation of cell growth (Wang et al. 2018).The anthocyanidins, identified in the SMAF, are water-soluble pigments that are stored in the vacuoles of plant cells. They are in all the organs of the plant and have positive effects on inflammatory states (innate immunity), related to their antioxidant and stimulating capacity of the immune system. The presence of these pigments increases lymphocyte proliferation and cytokine secretion (interleukin II) by the activated lymphocytes (Camacho et al. 2016).
In addition, other secondary metabolites found in SMAF, such as coumarins and reducing carbohydrates, constitute potent anticoagulants and bactericides against strains of Staphylococcus aureus and Escherichia coli (Escalona et al. 2016). The saponins detected, which are glycosides, widely distributed in plants, have antimicrobial and hypocholesterolemic effects (del Hierro et al. 2018). There was not reaction for bitter and astringent principles, although these properties have been related to the stimulation of gastric and bile juices. An excess causes a decrease in voluntary intake and decreases the productive behavior of animals (Han et al. 2018).
The presence of tannins in the SMAF could be beneficial for its antidiarrheal effects and for its performance as growth promoters in farm animals (Martínez et al. 2013). This polyphenolic metabolite has anti-inflammatory, vasoconstrictor, antioxidant, antibacterial and hypocholesterolemic properties (inhibits cholesterol absorption and expels it through faeces). However, excess tannins can limit the absorption of some nutrients, such as iron and proteins, as well as cause adverse intestinal processes (Pathak et al. 2016). Studies with Agave tequilana meal and SMAF as nutraceutical additives in pigs and birds diets positively modified the animal response (Iser et al. 2016a, Iser et al. 2016c and Chávez et al. 2019).
The quality indicators are mainly determined in the phytopharmaceuticals for humans (Shukla et al. 2018). However, although these analyzes are not common in animal foods, their evaluation is considered important. One of the international guidelines of nutraceuticals is the strict control of the quality of these products (Sociedad Española de Nutracéutica 2015), which certifies the high level of safety for their use as an additive in animal feeding.
As table 2 shows, the average pH of SMAF was approximately 5.18. This shows a certain inclination towards acidic compounds, which is due to a close relation with acidity. Madrigal and Sangronis (2007) found similar results when evaluating inulin and oligofructose (5 to 7), with slightly acidic values important for the stabilization and preservation of the product (Martínez et al. 2012).
Table 2 Quality indicators of the stems meal from Agave fourcroydes
| Indicators | Mean (n=3) | SD (±) | CV (%) |
|---|---|---|---|
| pH | 5.18 | 0.020 | 0.386 |
| Acidity (%) | 0.76 | 0.010 | 1.315 |
| Refractive index | 1.33 | 0.006 | 0.451 |
| Apparent density (g/mL) | 0.31 | 0.005 | 1.612 |
| Water soluble solids (0Brix) | 1.50 | 0.021 | 1.400 |
SD: standard deviation; CV: coefficient of variation
The refractive index and density constitute quick and simple tests to verify the purity degree and the percentage of solute dissolved in a given solution, as a critical control point (Shin et al. 2018). The refractive index showed an average value of 1.33, similar to other medicinal plants, such as the leaves of Anacardium occidentale (1.34) and Morinda citrifolia (1.35). This analysis in medicinal plants has been related to the greater presence of phytochemical compounds. In this regard, Torrenegra et al. (2015) indicated that a refractive index higher than 1.00 in essential oils of Minthostachys mollis is associated with secondary metabolites such as benzenic, aromatic and oxygenated terpenes. There were no reports that refer this quality parameter in the Agave spp., so this result is considered as a first for the proposed nutraceutical additive.
The low density of SMAF is related to the presentation of the product as meal, with high percent of dry matter (95.24%) (Iser et al. 2016b). The apparent density showed similar levels to those reported by Handreck et al. (2002), who recommend density up to 0.6 g/mL. Sánchez et al. (2015) reported inferior results to this study, when evaluating different agricultural substrates from A. tequilana. A similar response is reported in other organic substrates such as coconut fiber, rice husk and cane bagasse, with 0.077, 0.099 and 0.065 g/mL, respectively (Pire and Pereira 2003). However, the inulin extracted from Achicoria (1.35 g/mL) and Dhalia (1.19 g/mL) have higher apparent density (Campos et al. 2013). This indicator is directly related to the packing volume, which tends to be inversely proportional. In addition, generally, products derived from medicinal plants, such as aqueous extracts, fluids and tinctures, have a higher refractive index 1.00 (Rodríguez et al. 2012).
In relation to the soluble solids in the SMAF, there was a concentration of water-soluble active ingredients similar to Morinda citrifolia (1.57), which is convenient for direct use in diets and/or as phytopharmaceuticals. According to Koteswara et al. (2016), in products with low soluble solids content and high humidity values, the proliferation of pathogens is encouraged. Specifically, SMAF has high dry matter content (95.24 %) (Iser et al. 2016b) and beneficial water-soluble secondary metabolites, such as alkaloids, saponins, flavonoids, mucilages and reducing carbohydrates (table 1). This can positively influence on the quality of SMAF, as a future nutraceutical additive.
Table 3 shows that the organoleptic characteristics of the steams meal from Agave fourcroydes, stored for six months, is among the normal parameters for foods suitable for intake. A homogeneous powder was found, of good physical appearance and pleasant odor, without apparent presence of lumps. According to das Chagas et al. (2015), the lump is a distinctive feature of mycotoxin-contaminated products, which causes significant organoleptic changes.
Table 3 Organoleptic characteristics of the stems meal from Agave fourcroydes
| Indicators | Stems meal from Agave fourcroydes1 |
|---|---|
| Appearance | Good physical appearance (powder) |
| Dissolution capacity | It partially dissolves |
| Pulverization degree | Good pulverization |
| Color | Light beige |
| Odor | Nice characteristic of the product |
| Taste | Moderately sweet |
| Homogeneous capacity | Homogeneous powder |
1Professional experts from the Departamento de Química Analítica del Centro Provincial de Higiene y Epidemiología, Granma province, Cuba were used
The partial dilution and the moderately sweet taste of the product (SMAF) may be determined by the presence of fructans (oligofructose) and fructose (Iser et al. 2016b). Madrigal and Sangronis (2007) found that oligofructose has a moderately sweet taste and higher dilution than inulin, with a neutral taste. Also, Silos et al. (2011) reported that aguamiel from A. tequilana showed a sweet and pleasant taste.
In the SMAF there was a light beige color. According to Flores and Borredon (2013), the color depends on the characteristics of the product, these authors found a brown color in the dried Agave bagasse of salmiana species. In general, fructans have a color between white and pale yellow (Madrigal and Sangronis 2007); perhaps other plant nutrients could determine the color of the proposed product.
Conclusions
In the ether, ethanolic and aqueous extracts of SMAF, flavonoids, tannins, coumarins, anthocyanidins, reducing sugars and saponins predominated. This natural product could be used as a nutraceutical in the farm animals diets, due to the results of pH, acidity, refractive index, soluble solids, apparent density and organoleptic characteristics.
