UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO 
PROGRAMA DE MAESTRÍA Y DOCTORADO EN CIENCIAS DE LA PRODUCCIÓN Y DE LA SALUD 
ANIMAL 
FACULTAD DE ESTUDIOS SUPERIORES CUAUTITLÁN 
 
EVALUACIÓN DE BIOADSORBENTES EN LA PROTECCIÓN DE LA 
AFLATOXICOSIS MEDIANTE UN MODELO GASTROINTESTINAL IN VITRO 
DINÁMICO Y UN MODELO IN VIVO EN PAVOS (Meleagridis gallopavo) 
 
TESIS 
PARA OPTAR POR EL GRADO DE: 
DOCTORA EN CIENCIAS DE LA PRODUCCIÓN Y DE LA SALUD ANIMAL 
 
PRESENTA: 
MARÍA DE JESÚS NAVA RAMÍREZ 
 
TUTOR: 
DR. ABRAHAM MÉNDEZ ALBORES 
FACULTAD DE ESTUDIOS SUPERIORES CUAUTITLÁN 
 
COMITÉ TUTORAL: 
DR. CARLOS LÓPEZ COELLO 
UNAM-FACULTAD DE MEDICINA VETERINARIA Y ZOOTECNIA 
 
DR. GUILLERMO TÉLLEZ ISAÍAS 
UNIVERSIDAD DE ARKANSAS 
 
CUAUTITLÁN IZCALLI, ESTADO DE MÉXICO, 2024 
 
 
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Agradecimientos 
A la Universidad Nacional Autónoma de México y a la Facultad de Estudios Superiores 
Cuautitlán por permitirme ser parte de esta gran institución, donde he podido estudiar, 
aprender y enseñar, y que ha sido mi segundo hogar durante 16 años. 
A mi tutor, el Dr. Abraham por apoyarme, impulsarme y guiarme en un momento clave en mi 
vida. Gracias por enseñarme todo lo que soy profesionalmente durante estos últimos 7 años. 
Gracias por abrir las puertas del laboratorio 14 para que pudiéramos realizar todos los logros 
que hemos tenido hasta ahora.  
Al Dr. Carlos López por ser una persona que me apoyó y creyó en mí en los momentos que más 
lo necesité. Por sus sabios consejos que valoro y atesoro mucho.  
Al Dr. Guillermo por su apoyo incondicional que me bridó en todo momento, gracias por tanta 
generosidad y paciencia.   
A todos los doctores y doctoras que se involucraron el la supervisión y evaluación de este 
proyecto: Dra. Irma Candanosa, Dra. Patricia Ramírez, Dr. Ernesto Ávila, Dr. Sergio Gómez, Dr. 
Ignacio Soto y al Dr. Tórtora.  
Al programa de becas del Conahcyt por el apoyo otorgado a lo largo de mis estudios de 
posgrado. 
Investigación realizada con apoyo del Programa UNAM-PAPIIT IA101523. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dedicatorias 
A mi mamá, mis hermanas, mi sobrina y mi sobrino, que me han mostrado todo su cariño y 
apoyo de diversas maneras. Gracias por escucharme o leerme cuando lo más lo necesité. 
Todas ustedes son mi razón de ser.  
A mi esposa por ser mi gran apoyo de vida, por escucharme por horas y horas y por darme ese 
impulso para no tirar la toalla en los momentos más difíciles. Gracias por intentar aligerar y 
alegrar mis días con tanta atención y tanto amor.  
A todos mis gatitos que, sin saberlo, me han dado mucha alegría y amor.  
A Lis porque sin saberlo, ha sido mi mayor apoyo en todo este proceso. Gracias por ser esa 
amiga que tanto busqué.  
A Karla, Belén y Mari por las horas que me han dedicado en escuchar todas mis anécdotas y 
por darme los mejores consejos. Por mejorar mis días con las reuniones que solemos hacer. 
Por ser mis amigas desde que entramos a la UNAM…y lo que nos falta.  
A Sam, por ser esa persona con la que comparto situaciones y momentos parecidos. Por ser 
empática, sincera divertida y la persona más transparente que conozco.   
A Cecilia por ser mi amiga y compañera de posgrado. Por acompañarme y entenderme en todo 
este camino de maestría y doctorado. Por más etapas juntas. 
 
I 
 
Resumen 
La presente investigación se llevó a cabo en tres etapas: la primera consistió en un experimento in 
vitro, la segunda en un experimento in vivo y la tercera en un estudio de la microbiota intestinal de 
las aves. El experimento in vitro incluyó la producción y caracterización de cuatro adsorbentes a base 
de plantas ricas en clorofilas: espinaca (Spinacia oleracea), acelga (Beta vulgaris var. Cicla), perejil 
(Petroselinum crispum) y alfalfa (Medicago sativa). La caracterización incluyó a la microscopía 
electrónica de barrido, la espectroscopía de fluorescencia de rayos X, la difracción de rayos X, el 
potencial Z, el punto de carga cero, la espectroscopía de infrarrojo con transformada de Fourier y 
reflexión total atenuada, la espectroscopía UV-Vis y la espectroscopía de fluorescencia. El 
adsorbente de alfalfa obtuvo las mejores características para adsorber AFB1. Se evaluó su capacidad 
de adsorción utilizando dos modelos in vitro: uno pH-dependiente y otro que emuló el tracto 
gastrointestinal (TGI) de las aves, (250 ng AFB1/mL y 0.5% de adsorbente). El modelo pH-dependiente 
mostró una alta eficacia de adsorción (98.2% a pH 2, 99.9% a pH 5 y 98.2% a pH 7) en comparación 
con el modelo que emuló el TGI (88.8%). En el segundo experimento, el experimento in vivo, se 
utilizaron 350 pavitos Nicholas-700: (1) Control (libre de AFB1); (2) AF (250 ng AFB1/g); (3) Alfalfa 
(libre de AFB1+0.5% (p/p) de adsorbente); (4) AF+alfalfa (250 ng AFB1/g+0.5% (p/p) de adsorbente) 
y (5) AF+PCL (250 ng AFB1/g+0.5% (p/p) pared celular de levadura (PCL). En el grupo AF+alfalfa se 
observó el aumento del peso corporal y la ganancia de peso y la reducción del consumo de alimento. 
No obstante, el grupo Alfalfa mostró los mejores resultados en cuanto a los parámetros productivos 
y mejoró algunos niveles de la bioquímica sanguínea. Las lesiones histopatológicas en el hígado del 
grupo AF, mejoraron significativamente con la adición del adsorbente. En el tercer experimento, en 
el grupo Alfalfa se observó un aumento de bacterias benéficas (Faecalibacterium y Coprococcus 
catus) en el intestino, a diferencia del grupo AF, donde las bacterias oportunistas aumentaron 
(Streptococcus lutetiensis), provocando una disbacteriosis. En el grupo AF, la altura de las 
vellosidades intestinales disminuyó y la permeabilidad intestinal se vio afectada. Sin embargo, estos 
efectos se revirtieron con la adición del adsorbente de alfalfa. Los resultados indicaron que el 
adsorbente derivado de hojas de alfalfa utiliza mecanismos de adsorción electrostáticos, no 
electrostáticos y la formación de complejos clorofila-AFB1 para eliminar AFB1 en ambos modelos in 
vitro. En un modelo in vivo, la inclusión baja del adsorbente en el alimento contaminado con AFB1 
contrarrestó los efectos adversos de la toxina, y mejoró la microbiota del intestino. Estos hallazgos 
sugieren que las hojas de alfalfa podrían ser una alternativa adecuada como adsorbente de AFB1.  
Palabras clave: aflatoxina B1, alfalfa, adsorción, pavos, microbiota.  
 
II 
 
Abstract 
The present research was carried out in three stages: the first consisted of an in vitro experiment, 
the second in an in vivo experiment and the third in a study of the intestinal microbiota of birds. The 
in vitro experiment included the production and characterization of four adsorbents based on 
chlorophyll-rich plants: spinach (Spinacia oleracea), chard (Beta vulgaris var. Cicla), parsley 
(Petroselinum crispum) and alfalfa (Medicago sativa). The characterization included scanning 
electron microscopy, X-ray fluorescence spectroscopy, X-ray diffraction, zeta potential, point of zero 
charge, attenuated total reflectance-Fourier transform infrared spectroscopy, UV-Vis spectroscopy 
and fluorescence spectroscopy. The alfalfa adsorbent obtained the best characteristics to adsorb 
AFB1. Its adsorption capacity was evaluated using two in vitro models: one pH-dependent and 
another that emulated the gastrointestinal tract (GIT) of birds, (250 ng AFB1/mL and 0.5% 
adsorbent). The pH-dependent model showed a high adsorption efficiency (98.2% at pH 2, 99.9% at 
pH 5 and 98.2% at pH 7) compared to the model that emulated the GIT (88.8%). In the second 
experiment, the in vivo experiment, 350 Nicholas-700 poults were used: (1) Control (free of AFB1); 
(2) AF (250 ng AFB1/g); (3) Alfalfa (free of AFB1+0.5% (w/w) adsorbent); (4) AF+alfalfa (250 ng 
AFB1/g+0.5% (w/w) adsorbent) and (5) AF+YCW (250 ng AFB1/g+0.5% (w/w) yeast cell wall (YCW). In 
the AF+alfalfa group, an increase in body weight and weight gain and a reduction in feed 
consumption were observed. However, the Alfalfa group showed the best results in terms of 
production parameters and improved some levels of serum biochemistry. The histopathological 
lesions in the liver of the AF group improved significantly with the addition of the adsorbent. In the 
third experiment, an increase in beneficial bacteria (Faecalibacterium and Coprococcus catus) was 
observed in the intestine, unlike the group. AF group, where opportunistic bacteria increased 
(Streptococcus lutetiensis), causing dysbacteriosis. In the AF group, the height of the intestinal villi 
decreased and intestinal permeability was affected. However, these effects were reversed with the 
addition of the alfalfa adsorbent. The results indicated that the adsorbent derived from alfalfa leaves 
uses electrostatic, non-electrostatic adsorption mechanisms and the formation of chlorophyll-AFB1 
complexes to remove AFB1 in both in vitro models. In an in vivo model, low inclusion of the adsorbent 
in AFB1-contaminated feed counteracted the adverse effects of the toxin, and improved the gut 
microbiota. These findings suggest that alfalfa leaves could be a suitable alternative as an adsorbent 
for AFB1. 
Keywords: aflatoxin B1, alfalfa, adsorption, turkeys, microbiota. 
 
 
III 
 
Índice 
 
Resumen ............................................................................................................................................. I  
Abstract ............................................................................................................................................. II  
Índice ................................................................................................................................................ III  
Listado de figuras ………………………………………………………………………..……………………………..………………..IV 
Introducción ...................................................................................................................................... 1  
Antecedentes ..................................................................................................................................… 3  
Micotoxinas ........................................................................................................................................3 
Aflatoxinas ..........................................................................................................................................4 
La meleagricultura en México .............................................................................................................5 
Efecto de las aflatoxinas en los pavos.................................................................................................6 
Agentes adsorbentes...........................................................................................................................7 
Artículo 1 ..........................................................................................................................................10 
Artículo 2 ......................................................................................................................................... 28 
Artículo 3 ......................................................................................................................................... 37 
Discusión .......................................................................................................................................... 51 
Conclusiones .................................................................................................................................... 67 
Perspectivas…………………………………………………………………………………………………………………………………68 
Referencias .......................................................................................................................................69 
 
 
 
 
 
 
 
 
 
IV 
 
Listado de figuras 
Figura 1. Formación de enlaces de hidrógeno entre la molécula de AFB1 y los  
principales grupos funcionales existentes en el adsorbente de alfalfa en las tres  
secciones simuladas del tracto gastrointestinal de las aves (pH 2, 5 y 7)…………………………………………54 
Figura 2. Interacción electrostática de acuerdo con el pHpzc entre la superficie  
del adsorbente de hojas de alfalfa y los átomos de oxígeno de la  
molécula de AFB1 ………………………………………………………………………………………………………………………….56 
Figura 3. Interacción en la interfase del potencia ζ y el punto isoeléctrico (pI) entre  
el adsorbente de hojas de alfalfa y los átomos de oxígeno de  
la molécula de AFB1.…………………………………………………………………………………..………………………………...58 
Figura 4. Interacción entre la clorofila del adsorbente de las hojas de alfalfa y la  
molécula de la AFB1………………………………………………………………………………………………………………………59 
Figura 5. Efecto del adsorbente de alfalfa y la AFB1 sobre la microbiota intestinal,  
la permeabilidad intestinal, y la altura de las vellosidades intestinales de los pavos…………………..…..66 
 
 
1 
 
Introducción  
 
Las micotoxinas son compuestos químicos producidos por ciertos hongos filamentosos. Se han 
identificado alrededor de 400 micotoxinas potencialmente tóxicas; sin embargo, las toxinas que se 
consideran las más perjudiciales para la agricultura, la ganadería y la salud pública son los 
tricotecenos, las ocratoxinas, las aflatoxinas, la zearalenona, las fumonisinas, la patulina y la 
citrinina. 
A nivel mundial, las aflatoxinas (AF) son ampliamente reconocidas como las micotoxinas más 
relevantes en los alimentos tanto para humanos como para animales, debido a su toxigenicidad y 
su carácter ubicuo. Las AF de mayor a menor toxicidad son: la aflatoxina B1 (AFB1), la aflatoxina M1 
(AFM1), la aflatoxina G1 (AFG1), la aflatoxina M2 (AFM2), la aflatoxina B2 (AFB2), y la aflatoxina G2 
(AFG2). La AFB1 es conocida por su potencial hepatotóxico y carcinogénico, la cual puede contaminar 
una amplia variedad de semillas y cereales destinados al consumo de humanos y de animales, por 
lo que, puede causar importantes pérdidas económicas y problemas de seguridad alimentaria y de 
salud pública. 
La AFB1 conlleva una amplia variedad de efectos adversos en los animales, especialmente cuando 
se consume en dietas contaminadas, lo que puede desencadenar el desarrollo de aflatoxicosis, tanto 
en forma aguda como crónica. Algunos de los efectos más comunes en los animales incluyen: 
inmunosupresión, hepatotoxicidad, hepatocarcinogenicidad, mutagenicidad, anorexia, alteración 
de los parámetros productivos y de los valores bioquímicos y aumento de la morbilidad y la 
mortalidad. Las aves de producción, los cerdos y los bovinos son las especies domésticas que 
generan mayor preocupación económica en términos de aflatoxicosis. Sin embargo, las aves de 
producción son de las especies más susceptibles a las AF, especialmente los pavos debido a una 
eficiente bioactivación hepática mediada por el citocromo P450 y a una desintoxicación deficiente 
a través del glutatión S-transferasa (GST). 
Para reducir los efectos tóxicos que provoca la AFB1 en los pavos y mejorar la calidad, seguridad y 
disponibilidad de los alimentos en un contexto de crecimiento poblacional y cambios climáticos, se 
pueden implementar diversas estrategias basadas en métodos físicos, químicos y biológicos. En este 
contexto, la estrategia más utilizada en la industria avícola para el control y/o prevención de las AF 
es el método físico, principalmente la adición de materiales adsorbentes en el alimento. Los 
adsorbentes se clasifican en inorgánicos y orgánicos.  
2 
 
Los adsorbentes inorgánicos más usados son las bentonitas, las montmorillonitas, las esmectitas, 
las caolinitas y las zeolitas. Cabe señalar que, este tipo de adsorbentes inorgánicos tiene ciertas 
desventajas como la adsorción simultánea de diversos micronutrientes contenidos en la dieta de las 
aves, así como la liberación de dioxinas y metales pesados. Es por esta razón que, en los últimos 
años, se ha desarrollado y estudiado la eficacia de adsorción de la AFB1 de diversos adsorbentes 
orgánicos como el algarrobo, los tallos de uva micronizados, las hojas + bayas de Pyracantha 
koidzumii, el orujo de uva, algunas ligninas de plantas aromáticas, el polvo de Aloe vera, la cáscara 
de plátano, entre otros, los cuales se caracterizan por ser seguros, rentables y efectivos. Algunos 
estudios han demostrado que la inclusión de adsorbentes orgánicos que contienen un alto 
contenido de clorofilas (el kale, la lechuga y las hojas de Pyracantha koidzumii), son eficaces en la 
adsorción de AFB1.  
En consecuencia, la presente investigación tiene como objetivo conocer la naturaleza de la 
interacción entre cuatro nuevos adsorbentes orgánicos con un alto contenido de clorofilas y la 
molécula AFB1 empleando diversas técnicas de caracterización, de los cuales, sólo uno será 
seleccionado para evaluar su potencial de adsorción de AFB1 con un nivel bajo de inclusión, 
utilizando dos modelos in vitro (un modelo pH-dependiente y un modelo que emuló el TGI de las 
aves) y un modelo in vivo en pavos (Meleagridis gallopavo).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3 
 
Antecedentes  
 
Micotoxinas 
 
Algunas especies de hongos filamentosos, como Aspergillus, Fusarium, Penicillium, Claviceps y 
Alternaria, tienen la capacidad de producir metabolitos secundarios de bajo peso molecular que son 
altamente tóxicos, conocidos como micotoxinas (Huwig et al., 2001; Wang et al., 2020). Desde la 
década de 1960, se han identificado aproximadamente 400 tipos de micotoxinas, clasificadas en 
grupos según sus similitudes estructurales y efectos tóxicos (Wielogórska et al., 2016). Las 
micotoxinas son omnipresentes y se encuentran en una gran variedad de materiales, incluyendo 
alimentos para animales, alimentos para consumo humano, productos de origen animal y suelos 
(Tola y Kebede, 2016).  
Dado que en la mayoría de los países los cereales como el trigo, el maíz, la cebada, el centeno y la 
avena constituyen la principal fuente de nutrición, la Organización de las Naciones Unidas para la 
Alimentación y la Agricultura (FAO) estima que un porcentaje significativo de estos cereales está 
sujeto a la contaminación por micotoxinas, alcanzando aproximadamente el 25% de la producción 
mundial (Galvano et al., 2001; Sarrocco et al., 2019).  
Los hongos que generan micotoxinas en los alimentos se clasifican en dos categorías: aquellos que 
infectan antes de la cosecha, conocidos como hongos de campo, y los que emergen exclusivamente 
después de la cosecha, denominados hongos de almacenamiento (Atanda et al., 2011). Dentro de 
los hongos de campo, se identifican tres tipos: los fitopatógenos, como Fusarium graminearum 
(productor de deoxinivalenol y nivalenol); los que prosperan en plantas en etapa senescente o bajo 
estrés, como Fusarium moniliforme (generador de fumonisina), y ocasionalmente Aspergillus flavus 
(aflatoxina); y aquellos que inicialmente colonizan la planta antes de la cosecha, predisponiendo el 
producto a la contaminación por micotoxinas posterior a la recolección, tales como Penicillium 
verrucosum (ocratoxina) y A. flavus (aflatoxina) (Ayalew, 2010; Berthiller et al., 2013).  
 
 
 
 
 
 
4 
 
Aflatoxinas 
 
Producidas principalmente por los hongos del género Aspergillus, del cual, sólo 18 de sus especies 
pueden producir aflatoxinas: A. flavus, A. parasiticus, y A. nomius, A. aflatoxiformans, A. 
arachidicola, A. austwickii, A. cerealis, A. pseudotamarii, A. sergii, A. bombycis, por mencionar 
algunas (Bennett y Klich, 2003; Frisvad et al., 2019). 
Los períodos de sequía, combinados con altas temperaturas, incrementan notablemente la 
producción de aflatoxinas en el campo; además, las aflatoxinas se sintetizan exponencialmente 
durante el almacenamiento de varios meses en condiciones de calor y alta humedad (Sanders et al., 
1993; Villers, 2014). La temperatura mínima requerida para la producción de aflatoxinas oscila entre 
10 y 12°C, con una temperatura óptima entre 27 y 30°C, y una máxima de 40 a 42°C (Frisvad, 2012; 
Milani, 2013). Los hongos suelen proliferar en un rango de pH de 4 a 8 y a niveles de actividad de 
agua (aw) superiores a 0.73 (Vujanovic et al., 2001). La humedad también desempeña un papel 
crucial en el crecimiento del hongo y la subsiguiente producción de aflatoxinas en los granos durante 
su almacenamiento. Cuando la humedad relativa supera el 65%, Aspergillus puede desarrollarse sin 
dificultades (Villers, 2014). 
Las principales aflatoxinas se dividen en grupos B (azul) y G (verde), debido a su fluorescencia azul o 
verde cuando se exponen a la luz ultravioleta (λ = 365 nm). Los subíndices 1 y 2 indican la movilidad 
relativa de las aflatoxinas en la cromatografía de capa fina, basada en su peso molecular (Carvajal, 
2013). El orden de toxicidad de las aflatoxinas, de mayor a menor, es el siguiente: AFB1, AFM1, AFG1, 
AFM2, AFB2 y AFG2, siendo la AFB1 la aflatoxina más prevalente en productos alimenticios, seguida 
por la AFG1. Las aflatoxinas B2 y G2 son biológicamente inactivas, a menos que se oxiden 
metabólicamente a AFB1 y AFG1 (Bbosa et al., 2013; Murugesan et al., 2015; Neeff et al., 2013). La 
aflatoxina M1 y la aflatoxina M2 son metabolitos hidroxilados de AFB1 y AFB2, respectivamente (Lee 
et al., 2014). 
El peso molecular de la AFB1, AFB2, AFG1 y AFG2 es de 312, 314, 328 y 330, respectivamente (Mousavi 
et al., 2018). La estructura de las aflatoxinas está formada por un núcleo cumarínico y otro difurano 
el cual ocasiona que la molécula tenga mayor rigidez. Las aflatoxinas B1 y G1 tienen un enlace 
insaturado en la posición 8,9 en el anillo terminal de furano (Miranda et al., 2013). La AFB1 se 
presenta como un sólido cristalino de color blanco pálido a amarillo, con ausencia de olor. Es soluble 
en varios disolventes orgánicos, tales como cloroformo, metanol, acetonitrilo y acetona (Sirhan et 
al., 2014). La AFB1 es muy estable a temperaturas superiores a 100 °C, se puede lograr poca o casi 
5 
 
ninguna descomposición (Campagnollo et al., 2016), aunque se pueden destruir en autoclave con 
amonio o hipoclorito de sodio (Carvajal, 2013). 
La AFB1 se encuentra en diversos cereales, semillas oleaginosas, especias y frutos secos. Se ha 
informado que los cereales más susceptibles a la contaminación por aflatoxinas incluyen la cebada, 
el maíz, el arroz, el sorgo y el trigo, así como las semillas oleaginosas como el algodón, el maní los 
girasoles y los cacahuetes (Bryden, 2012). 
El consumo de cereales contaminados con AF y productos a base de cereales como alimento 
destinado a consumo humano y animal, se considera un problema grave que debe controlarse 
mediante la implementación de estrategias de prevención y legislación (Mousavi et al., 2018). En 
México, existe la Norma Oficial Mexicana (NOM-188-SSA1-2010, Productos y servicios. Control de 
aflatoxinas en cereales para consumo humano y animal. Es de cumplimiento obligatorio en el 
territorio nacional y establece el límite máximo permitido de aflatoxinas en los cereales destinados 
al consumo animal, así como las normativas para su adecuado transporte y almacenamiento. En el 
Apéndice A, que establece los límites permitidos para el consumo animal, se especifica que los 
cereales con un contenido de aflatoxinas superior a 20 µg/kg serán destinados al consumo animal 
(NOM-188-SSA1-2010). 
 
La meleagricultura en México 
 
El guajolote (Meleagridis gallopavo), es una especie autóctona y de gran importancia cultural para 
las zonas rurales de México, la cría y producción de pavo es una actividad económica que desempeña 
un papel crucial en la creación de empleos directos e indirectos, así como en la preservación de las 
tradiciones socioculturales en el Estado de México. Sin embargo, la producción de pavos es baja y 
principalmente se destina al autoconsumo mediante la elaboración de platillos tradicionales. En 
menor medida, tanto el pavo como la carne se venden en los mercados locales para mejorar los 
ingresos familiares (Rodríguez-Licea et al., 2019). 
El pavo se posiciona como la segunda especie avícola más relevante a nivel nacional, representando 
el 0.2% de la producción pecuaria mexicana en la industria avícola durante el año 2021 (UNA, 2023). 
Esta posición se debe a su capacidad de adaptación a una amplia variedad de climas y sistemas de 
producción, destacándose especialmente en los sistemas de producción en pequeña escala (Cruz-
Lujan et al., 2023). En el año 2021, la producción de pavo alcanzó las 8,406 toneladas, generando un 
valor de 1,109 millones de pesos (UNA, 2023). 
6 
 
México ha mantenido una tradición histórica en la crianza de pavos, siendo los estados de Yucatán, 
Puebla y el Estado de México los principales productores, contribuyendo conjuntamente con el 59% 
de la producción nacional. Esta actividad guarda una estrecha relación con la producción agrícola, 
ya que suministra granos de cereales como trigo, avena, sorgo y maíz principalmente, los cuales son 
fundamentales para la alimentación de estas aves (Rodríguez-Licea et al., 2019). 
 
Efecto de las aflatoxinas en los pavos 
 
La extrema sensibilidad de aves de producción a AFB1 ha sido bien documentado, antes de identificar 
el agente etiológico, en Inglaterra a principios de 1960, se observaron por primera vez los efectos 
tóxicos de la AFB1 en más de 100,000 pavos, lo que condujo al surgimiento de la denominada 
“Enfermedad X de los pavos” (Quist et al., 2000).  
Los pavos exhibieron signos de intoxicación extrema, como opistótonos (rigidez anormal de los 
músculos), enteritis y una tasa de mortalidad del 100%. Los estudios post-mortem revelaron lesiones 
de inflamación intestinal severa, junto con hallazgos histológicos que mostraron necrosis hepática 
aguda, hiperplasia de los conductos biliares y lipidosis hepática. Pronto se determinó que los pavos 
habían sido alimentados con harina de cacahuate contaminada con A. flavus, importada desde 
Brasil, lo que se asoció directamente al desarrollo de esta patología (Do y Choi, 2007; Rushing y 
Selim, 2019). Desde entonces se ha comprobado que los pavos son más susceptibles que los pollos 
a los efectos tóxicos de la AFB1 en su dieta (Giambrone et al., 1985). Los pavos afectados por 
aflatoxinas típicamente muestran signos como inapetencia, reducción de la actividad, marcha 
inestable, rendimiento deficiente, disminución en el peso de los órganos, anemia, inmunosupresión, 
alta morbilidad y, en casos graves, la muerte (Klein et al., 2020). Durante la necropsia, se observa 
una condición corporal generalmente buena, pero con signos de congestión y edema generalizados. 
Las aflatoxinas presentes en los alimentos contaminados se absorben rápidamente en el intestino 
delgado, afectando principalmente al hígado y provocando trastornos metabólicos. El hígado suele 
presentar congestión, firmeza, hemorragia, necrosis, cambios grasos en los hepatocitos y 
hepatomegalia. Los cambios microscópicos del hígado incluyen la degeneración hidrópica, 
hiperplasia de conductos biliares, y fibrosis periportal (Magnoli et al., 2011). La degeneración grasa 
y la proliferación de los conductos biliares, por lo general, se manifiestan como un aumento en la 
actividad de las enzimas hepáticas, trastornos de la coagulación y una reducción en la producción 
de proteínas (Fernández et al., 1995).  
7 
 
Agentes adsorbentes 
 
Ante estos problemas globales, numerosas organizaciones, instituciones, productores e 
investigadores se han comprometido a desarrollar diversas estrategias y prácticas de prevención y 
descontaminación. El objetivo es reducir los riesgos asociados con las AF en los alimentos. Las 
estrategias de control y prevención de micotoxinas abarcan acciones tanto en la etapa de 
precosecha como en la de poscosecha (Huwig et al., 2001; Ringot et al., 2007). Sin embargo, cuando 
la prevención en el campo o durante la cosecha no es suficiente, es necesario recurrir a 
procedimientos de descontaminación de micotoxinas mediante métodos físicos, químicos o 
biológicos. En la actualidad, los mayores esfuerzos se han centrado en eliminar o reducir el efecto 
de las micotoxinas mediante el uso de diversos productos que pueden suprimir o disminuir su 
absorción, promover la excreción o modificar su modo de acción. Estas sustancias han sido 
reconocidas por la Comisión Europea (EC) como agentes desintoxicantes (Denli y Pérez, 2006; Vila-
Donat et al., 2018). Dichos agentes desintoxicantes se dividen en agentes adsorbentes y agentes 
biotransformadores. Los agentes adsorbentes es uno de los métodos más prácticos para la 
descontaminación de AF dentro de la industria animal (Magnoli et al., 2011; Vijayanandraj et al., 
2014).  
Los adsorbentes de micotoxinas son compuestos de alto peso molecular con la capacidad de unir a 
las micotoxinas. Su modo de acción radica en evitar la absorción de la AFB1 presente en el alimento 
al no disociarse en el tracto gastrointestinal (TGI) del animal después de su ingestión. Esto limita su 
biodisponibilidad y, por ende, reduce la exposición a la AFB1, previniendo así sus efectos 
hepatotóxicos (Vila-Donat et al., 2018; Rushing y Selim, 2019). Los adsorbentes se dividen en 
adsorbentes orgánicos y adsorbentes inorgánicos (Vila-Donat et al., 2018). Los adsorbentes 
inorgánicos son los más utilizados en la industria avícola, incluyendo a los aluminosilicatos 
(tectosilicatos y filosilicatos), a los aluminosilicatos de calcio y sodio hidratado (HSCAS), a las arcillas 
de esmectita, a las zeolitas y a las clinoptilolitas. Se ha reportado que las arcillas también son capaces 
de adsorber micronutrientes de la dieta animal y tienen efectos negativos en la disponibilidad de 
minerales y oligoelementos. Además, existe el riesgo de que las arcillas naturales se contaminen con 
dioxinas y metales pesados (Vila-Donat et al., 2018). 
Ante las relativas desventajas de los adsorbentes inorgánicos, en los últimos años se ha propuesto 
el desarrollo de adsorbentes orgánicos, ya que son seguros, rentables y efectivos (Avantaggiato et 
al., 2014; Ringot et al., 2007). 
8 
 
Diversos adsorbentes orgánicos, como fibras de plantas, residuos agrícolas, extractos de paredes 
celulares de levadura y bacterias, han demostrado ser prometedores en su capacidad de adsorción 
de AFB1, tanto en modelos in vitro como en modelos in vivo. Sin embargo, pocos de estos han sido 
investigados en modelos in vivo (Bueno et al., 2001). Los materiales como la paja, las cáscaras de 
nueces y almendras, así como las semillas, la pulpa de frutas, los materiales vegetales, las plantas, 
los cereales y forrajes, están compuestos principalmente de celulosa, lignina, hemicelulosa, pectina 
y lípidos, entre otros compuestos. Además, los principales componentes de las plantas son los 
fenólicos, flavonoides, cumarinas, clorofila y clorofilina, los cuales poseen propiedades 
quimioprotectoras contra compuestos cancerígenos como la AFB1 (Boudergue et al., 2009). Los 
diversos compuestos de los adsorbentes orgánicos contienen diferentes grupos funcionales, los 
cuales son los responsables de la unión con la AFB1 (Bočarov-Stančić et al., 2018; Vila-Donat et al., 
2018). 
Algunos estudios reportan porcentajes altos de adsorción de aflatoxinas con el uso de adsorbentes 
orgánicos como: el orujo de uva (82%), la cáscara de almendra (87%), los tallos y las hojas de la 
alcachofa (55%), el algarrobo (100%), el orujo de oliva (74%), los tallos de uva micronizados (96%), 
las hojas+bayas de Pyracantha koidzumii (82%), la piel de durian (99%), el kale (94%), la lechuga 
(95%), la cola de caballo (71%) y algunas ligninas de plantas aromáticas (80%) (Avantaggiato et al., 
2014; Greco et al., 2019; Fernandes et al., 2019; Karmanov et al., 2020; Ramales-Valderrama et al., 
2016). En consecuencia, debido a que los adsorbentes orgánicos han sido considerados como una 
alternativa importante para la reducción o remoción de las aflatoxinas, la presente investigación 
tiene como objetivo principal estudiar la eficacia de cuatro nuevos adsorbentes orgánicos, con un 
alto contenido en clorofilas contra la remoción de AFB1 en dos modelos in vitro y un modelo in vivo. 
 
9 
 
Artículos científicos 
 
Artículo 1. Removal of Aflatoxin B1 Using Alfalfa Leaves as an Adsorbent Material: A Comparison 
between Two In Vitro Experimental Models. 
 
Artículo 2. Efficacy of powdered alfalfa leaves to ameliorate the toxic effects of aflatoxin B1 in 
turkey poults. 
 
Artículo 3. Exploring the effects of an alfalfa leaf-derived adsorbent on microbial community, ileal 
morphology, barrier function, and immunity in turkey poults during chronic aflatoxin B1 exposure.  
 
Citation: Nava-Ramírez, M.d.J.;
Vázquez-Durán, A.;
Figueroa-Cárdenas, J.d.D.;
Hernández-Patlán, D.; Solís-Cruz, B.;
Téllez-Isaías, G.; López-Coello, C.;
Méndez-Albores, A. Removal of
Aflatoxin B1 Using Alfalfa Leaves as
an Adsorbent Material: A
Comparison between Two In Vitro
Experimental Models. Toxins 2023, 15,
604. https://doi.org/10.3390/
toxins15100604
Received: 8 September 2023
Revised: 1 October 2023
Accepted: 3 October 2023
Published: 8 October 2023
Copyright: © 2023 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/).
toxins
Article
Removal of Aflatoxin B1 Using Alfalfa Leaves as an Adsorbent
Material: A Comparison between Two In Vitro
Experimental Models
María de Jesús Nava-Ramírez 1 , Alma Vázquez-Durán 1, Juan de Dios Figueroa-Cárdenas 2,
Daniel Hernández-Patlán 3 , Bruno Solís-Cruz 3 , Guillermo Téllez-Isaías 4 , Carlos López-Coello 5
and Abraham Méndez-Albores 1,*
1 Unidad de Investigación Multidisciplinaria (UIM) L14 (Alimentos, Micotoxinas y Micotoxicosis), Facultad de
Estudios Superiores Cuautitlán (FES-C), Universidad Nacional Autónoma de México (UNAM),
Cuautitlán Izcalli 54714, Mexico; mari_551293@comunidad.unam.mx (M.d.J.N.-R.);
almavazquez@comunidad.unam.mx (A.V.-D.)
2 Cinvestav-IPN Unidad de Querétaro, Libramiento Norponiente No. 2000, Fraccionamiento Real de Juriquilla,
Queretaro 76230, Mexico; jfigueroa@cinvestav.mx
3 UIM L5, FES-C, UNAM, Mexico City 54714, Mexico; danielpatlan@comunidad.unam.mx (D.H.-P.);
bruno_sc@comunidad.unam.mx (B.S.-C.)
4 Division of Agriculture, Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA;
gtellez@uark.edu
5 Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia, UNAM,
Mexico City 04510, Mexico; coelloca@unam.mx
* Correspondence: albores@unam.mx
Abstract: An adsorbent material derived from alfalfa leaves was prepared and further characterized,
and its efficacy for removing aflatoxin B1 (AFB1) was investigated. Characterization consisted of the
use of attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), environmen-
tal scanning electron microscopy (ESEM), X-ray fluorescence spectroscopy (XRF), X-ray diffraction
(XRD), point of zero charge (pHpzc), zeta potential (ζ-potential), UV-Vis diffuse reflectance spec-
troscopy, and spectral analysis. To determine the adsorption capacity against AFB1 (250 ng AFB1/mL),
pH-dependent and avian intestinal in vitro models were used. The adsorbent inclusion percentage
was 0.5% (w/w). In general, the pH-dependent model gave adsorption percentages of 98.2%, 99.9%,
and 98.2%, evaluated at pH values of 2, 5, and 7, respectively. However, when the avian intestinal
model was used, it was observed that the adsorption percentage of AFB1 significantly decreased
(88.8%). Based on the characterization results, it is proposed that electrostatic, non-electrostatic, and
the formation of chlorophyll-AFB1 complexes were the main mechanisms for AFB1 adsorption. From
these results, it can be concluded that the adsorbent derived from alfalfa leaves could be used as an
effective material for removing AFB1 in in vitro digestion models that mimic the physiological reality.
Keywords: aflatoxin B1; alfalfa leaves; adsorption; in vitro digestion models; characterization
Key Contribution: An unmodified adsorbent prepared from alfalfa leaves was utilized to remove
the carcinogen AFB1 in two experimental models. In the pH-dependent model, it was determined
that pH did not affect AFB1 uptake, yielding AFB1 adsorption values up to 98%. However, when the
avian intestinal model was used, a moderate reduction in the AFB1 adsorption (88.8%) was attained.
Overall, the adsorbent material showed a significant ability to remove AFB1 in in vitro digestion
models that mimic the in vivo reality.
1. Introduction
Mycotoxins are toxic secondary metabolites synthesized by several species of fila-
mentous fungi [1]. Until now, more than 300 types of mycotoxins have been known [2],
Toxins 2023, 15, 604. https://doi.org/10.3390/toxins15100604 https://www.mdpi.com/journal/toxins
Toxins 2023, 15, 604 2 of 18
including aflatoxin B1 (AFB1), which is considered one of the most toxic substances because
of its highly carcinogenic potential to humans and animals [3,4]. The International Agency
for Research on Cancer has classified aflatoxin as a group 1 carcinogen [5]. In general, AFB1
is considered a natural contaminant of various agricultural products intended for feed
preparation, and the consumption of these contaminated products causes serious health
problems; therefore, there are considerable economic losses in the poultry industry due to
aflatoxin consumption [6,7].
To reduce the negative effects produced by aflatoxins, various strategies have been
proposed; one of the most promising and used in the feed industry is the addition of
adsorbent materials [8,9]. Adsorbents are compounds that are characterized by having a
large molecular weight; consequently, AFB1 present in the contaminated feed is capable of
binding to these materials without dissociating throughout the gastrointestinal tract (GIT)
of the bird, limiting AFB1 absorption and promoting its elimination via the feces [8,9]. The
most widely used mycotoxin adsorbents are inorganics such as zeolites, aluminosilicates,
hydrated calcium and sodium aluminosilicate, and clays; however, they are mostly non-
biodegradable compounds, and some of them release toxic components such as heavy
metals and dioxins, in addition to indiscriminately adsorbing some nutrients from the
diet [8,10]. In recent years, various plant-based adsorbents have been developed, which
offer an efficient, economical, and environmentally friendly alternative to remove AFB1,
in addition to maintaining the nutritional value of the diet [11]. In the scientific literature,
some of the agrisorbents that have been already tested to remove AFB1 are based on grape
and olive (pomaces, seeds, and stems), banana peel, Formosa firethorn (leaves and berries),
lignins, micronized fibers, Aloe vera, lettuce, field horsetail, kale, durian peel, blueberry
pomace, artichoke wastes, and almond hulls [12,13].
Due to the integral benefits that characterize a plant-based adsorbent, more research
is required to evaluate the adsorption potential against AFB1 of other materials that are
destined to be consumed with the feed. Alfalfa (Medicago sativa L.) is a forage used
worldwide in animal feed due to its adaptability, high protein content, and low production
cost [14]. The values of crude protein, crude fiber, crude cellulose, and metabolizable
energy contained in alfalfa are 17.5%, 24.1%, 20%, and 1200 kcal/kg, respectively [14,15].
Little is known about the effects of fresh forage consumption by poultry [16]. For instance,
Suwignyo et al. [17] reported that fresh alfalfa supplementation in ducks affected feed
intake, body weight gain, and feed conversion ratio. However, it has been reported that
alfalfa meal can be used in poultry diets due to its high nutritional and pigment content,
lower amount of cellulose, and higher digestibility [14]. Other authors have reported
that the inclusion of powdered alfalfa in poultry diets resulted in positive effects such
as a reduction in the feed conversion ratio, mortality, abdominal fat, and cholesterol
content of the yolk. In addition, powdered alfalfa increased the content of the pectoral
muscle and body weight as well as improved the height of the villi and depth of the
duodenal crypts [18,19]. In general, there is a considerable variation in the recommended
levels of inclusion of alfalfa in poultry diets. Suwignyo et al. [17], Shahsavari et al. [20],
and Suwignyo et al. [18] recommended an inclusion limit of 6%, 5%, and 3% (w/w),
respectively. However, alfalfa has a high nutritional value as it is a good source of proteins,
minerals, vitamins, flavonoids, and isoflavones [18]. In addition, alfalfa is a natural source
of pigments such as xanthophylls, chlorophylls, and carotenoids, giving poultry carcasses
a desirable yellowish color [15,18]. In this research, we hypothesize that the use of alfalfa
as an adsorbent material could provide certain advantages in the poultry industry, since
alfalfa can have a dual-purpose role, that is, as an AFB1 adsorbent and as a feed supplement
due to its large amount of nutrients and phytochemicals. To the best of our knowledge,
there are no studies on the use of alfalfa leaves as an AFB1 adsorbent; therefore, the
aim of the present study was to prepare and characterize an adsorbent material derived
from alfalfa leaves and investigate its AFB1 adsorption capacity in two experimental
in vitro models.
Toxins 2023, 15, 604 3 of 18
2. Results and Discussion
2.1. Adsorption Experiments
2.1.1. The pH-Dependent Model
The pH is an important factor in adsorption experiments; in most cases, pH can affect
the surface charge of the functional groups of the adsorbent, as well as the ionization of
the toxin [21]. For this reason, an in vitro study was carried out with three different pH
values (2, 5, and 7) to evaluate the adsorption capacity of the plant-based adsorbent and the
YCW (reference material), using a 0.5% (w/v) inclusion rate. Figure 1 shows that the pH
variation had no influence on the removal of AFB1 by the alfalfa adsorbent; consequently,
there was no statistical significance in the percentage of AFB1 adsorption at the three tested
pH values. In general, the percentage of adsorption of AFB1 with the adsorbent derived
from alfalfa leaves was significantly higher at the three pH values compared to the YCW. At
pH values of 2, 5, and 7, the adsorbent prepared from alfalfa leaves removed 98.2 ± 0.4%,
99.9 ± 0.2%, and 98.2 ± 2.9% of the toxin, respectively. On the contrary, the YCW adsorbed
17.4 ± 3.9%, 63.5 ± 2.5%, and 65.4 ± 6.2% of the mycotoxin, respectively. Therefore, the
adsorbent prepared from alfalfa leaves removed 80.2%, 36.5%, and 33.5% more AFB1 than
YCW at pH 2, 5, and 7, respectively. The control (without the addition of adsorbent) showed
a considerable deficiency of AFB1 uptake (<5%).
ff
ff
tt
ff
ff
ffi
tt
ffi tt ffi
0
20
40
60
80
100
NS
a
a
ControlAlfalfa
 
AF
B 1 a
ds
or
pt
io
n 
(%
)
 
pH 7pH 2 pH 5 pH 7pH 2 pH 5 pH 7pH 2 pH 5
YCW
b
NS
Figure 1. The AFB1 adsorption capacity of the adsorbent material derived from alfalfa leaves and
the yeast cell wall (YCW) using a pH-dependent in vitro model. Boxes and whiskers not sharing a
common superscript differ significantly (Tukey test p < 0.05). NS = not significant.
In general, the charge of the AFB1 molecule depends on its acid dissociation constant
(pKa = 17.79); thus, AFB1 is neither protonated nor deprotonated within the pH range
of this research. Consequently, the variation in the pH of the medium did not affect the
adsorption of AFB1 regardless of the surface charge of the adsorbent. These findings are in
accordance with the results of other authors [11,22–25].
2.1.2. The Avian Intestinal Model
Another in vitro digestive model was also used, which aimed to simulate the con-
ditions of the GIT of birds. Figure 2 shows the percentage of AFB1 adsorption. In the
intestinal section, the adsorbent derived from alfalfa leaves had a significantly higher per-
centage of adsorption (88.8 ± 4.1%) compared to the YCW (33.6 ± 3.1%). On the contrary,
the control group (without adsorbent addition) had a marked lack of AFB1 adsorption
(<3%). Various authors have performed AFB1 adsorption studies under the simulation
of some GIT conditions; for example, Adunphatcharaphon et al. [26] carried out a study
in a standardized digestion model, which included the phases of oral, gastric, and small
intestine digestion. The authors using acid-treated durian peel showed that the adsorbent
had 98.4% aflatoxin uptake. Moreover, Vázquez-Durán et al. [27] carried out an in vitro
Toxins 2023, 15, 604 4 of 18
digestive model simulating the GIT conditions of birds. In the study, adsorbents prepared
from kale and lettuce removed 93.6% and 83.7% of the mycotoxin, respectively. In the
present investigation, a clear decrease in the adsorption capacity of AFB1 was observed
with the adsorbent derived from alfalfa leaves in the avian intestinal model compared to
the pH-dependent model. The adsorption of AFB1 in the avian intestinal model was mainly
affected by the lack of interaction between the adsorbent and the AFB1 because the feed
matrix did not allow the encounter between the adsorbent and the adsorbate, in addition
to the effect exerted by the gastric enzymes. In this context, Rasheed et al. [13] compared
the efficacy of blueberry pomace (BB) to remove AFB1 using a static in vitro model and a
model that simulated gastric conditions. The authors reported that the in vitro model at
pH 3 had better adsorption capacity compared to the model that simulated gastric con-
ditions. This decrease in the BB adsorbent efficiency was attributed to the difficulty of
trapping the AFB1 molecule due to the presence of pepsin in the model that simulated the
gastric fluid. These results are consistent with our findings.
Control
Alfalfa
YCW
0 20 40 60 80 100
 
c
AFB1 adsorption (%)
a
b
ff
−
− −
−
−
−
− −
−
−
−
− −
Figure 2. The AFB1 adsorption capacity of the adsorbent material derived from alfalfa leaves and the
yeast cell wall (YCW) using the avian intestinal model. a,b,c Mean values ± standard error. Means
not sharing a common superscript differ significantly (Tukey p < 0.05).
2.2. Characterization
2.2.1. FTIR-ATR
The FTIR-ATR spectra of the adsorbent material derived from alfalfa leaves and the
YCW were collected in the spectral range of 4000 to 400 cm−1 (Figure 3). Table 1 compiles
the primary active FTIR vibrations and their functional groups. In general, the functional
groups present in the adsorbent materials were further analyzed to investigate the possible
interactions between the AFB1 molecule and the functional groups [28]. The adsorbent
derived from alfalfa leaves (Figure 3a) exhibited a high intensity of the hydroxyl group at
3668 and 3280 cm−1, the methyl group at 2964, 2917, and 2850 cm−1, the vibration associated
with the carboxyl group at 1599 cm−1, the strong vibration of aromatic compounds at
1408 and 1242 cm−1, and the strong molecular vibration of the C–O bond at approximately
1066 cm−1 [13,27,29–31]. On the other hand, the main bands related to chlorophylls are the
C–O stretching, the C–C stretching vibration, and the bands originated from the stretching
of the methyl groups [32,33]. As can be seen, both adsorbent materials mainly contained
four functional groups: hydroxyl at 3281 cm−1, aliphatic chains at 2923 and 2853 cm−1,
aromatic compounds at 1532, 1455, 1369, and 1244 cm−1, and the carbonyl group at
approximately 1025 cm−1. Furthermore, in Figure 3b, it is also possible to distinguish
three characteristic regions of the YCW adsorbent, which correspond to polysaccharides
(1182–842 cm−1), proteins (1573–1701 cm−1), and lipids (2797–2990 cm−1) [34].
Toxins 2023, 15, 604 5 of 18
4000 3500 3000 2500 2000 1500 1000 500
40
60
80
100
4000 3500 3000 2500 2000 1500 1000 500
40
60
80
100
(b)
Tr
an
sm
itt
an
ce
 (%
)
Wavenumber (cm−1)
US
N
M
K
H
F
E
D
CB
A
(a)
Tr
an
sm
itt
an
ce
 (%
)
Wavenumber (cm−1)
RTM
F V
Q
O
LJI
GE
D
B
P
−
β β
β β →
α β α
α β α
α β α
Figure 3. Fourier transform infrared spectroscopy (FTIR) spectrum of (a) the adsorbent material
derived from alfalfa leaves and (b) the yeast cell wall (YCW).
Table 1. Bands assignments and functional groups in the adsorbent material derived from alfalfa
leaves and the yeast cell wall (YCW).
Band
Wavenumber (cm−1)
Functional Group
Alfalfa YCW
A 3668 NF O–H stretching
B 3280 3281
O–H and N–H stretching vibrations
(carbohydrate and protein)
C 2964 NF CH2 antisymmetric stretching (lipids)
D 2917 2923 –(CH2)n– antisymmetric stretching (lipids)
E 2850 2853 C–CH3 symmetric stretching (lipids)
F 1732 1710 C=O stretching (phospholipid esters)
G NF 1629 Amide I (N–H bending and C=O stretching)
H 1599 NF COOR (carboxylate group)
I NF 1532 Amide II (C–N stretching and N–H bending)
J NF 1455 OH bending vibration in carboxylic acids
K 1408 NF –CH2 deformation (cellulose)
L NF 1369 β-anomeric carbons (β-glucans)
M 1242 1244
PO2
− antisymmetric stretching (DNA, RNA,
phospholipid, phosphorylated protein)
N 1066 NF
C–O stretching (carbohydrate)jialiC–O–P
stretching (phosphate ester)
O NF 1025 C–O stretching (carbohydrates)
P NF 887 β-anomeric carbons β (1→3)-glucans
Q NF 812
Mannans (C–O–C, C–C, and C–OH stretching of
pyranose ring)
R NF 670 Polysaccharides (α- and β-glucans, α-mannan)
S 611 NF NH2 wag (primary amines)
T NF 575 Polysaccharides (α- and β-glucans, α-mannan)
U 534 NF In plane and out-of-plane ring deformations
V NF 508 Polysaccharides (α- and β-glucans, α-mannan)
YCW = yeast cell wall. NF = not found.
According to what was reported by Vázquez-Durán et al. [27] and Shar et al. [29],
the functional groups present in the adsorbents (hydroxyl, methyl, carboxyl, aromatic,
and carbonyl) contribute to the adsorption of AFB1. Bearing this in mind and that the
alfalfa and YCW share most of the vibrations of these functional groups, the calculation
of the bond indexes was carried out to know the real amount of each functional group.
According to the results (Figure 4), the adsorbent prepared from alfalfa leaves contained
Toxins 2023, 15, 604 6 of 18
fewer methyl groups compared to the YCW; however, in terms of aromatic groups, there
was no statistical significance between the two tested materials. On the other hand, the
YCW contained 1.9 and 2.5 times more hydroxyl and carbonyl groups, compared to the
adsorbent derived from alfalfa leaves. It has been reported that the adsorption of AFB1 can
occur due to the hydrophobicity of the adsorbents, and this hydrophobicity is conferred
to its surface by the presence of hydrophobic groups such as methyl and aromatics; on
the contrary, if hydrophilic groups are present (hydroxyl, carboxyl, and carbonyl), the
adsorption efficiency of the adsorbents is compromised [27]. It is important to note that
the spectrum of the YCW lacks carboxyl groups compared to the adsorbent derived from
alfalfa leaves, which shows a prominent band at around 1599 cm−1. It is well known
that functional groups deprotonate if they are at a pH higher than their pKa [35]. In this
context, the pKa of the carboxyl group is ~4.5; thus, from pH values above 4.5 the carboxyl
loses the hydrogen atom; consequently, this functional group is only capable of forming
hydrogen bonds with the oxygen atoms of the AFB1 molecule at pH of 2 (the proventriculus
environment). Furthermore, the pKa of the hydroxyl group is ~11.6, the amine group is
~40, and the amide group is ~18; therefore, the presence of these three functional groups
favors the formation of hydrogen bonds with the oxygen atoms of the AFB1 molecule in the
three simulated sections (crop, proventriculus, and intestine). Regarding the YCW, several
authors have reported that β-d-glucans, glucomannans, and mannan-oligosaccharides are
the main components responsible for the mycotoxin adsorption [36].
ffi
−
β
ff
Figure 4. Bond indexes of the main functional groups of the adsorbent materials. a,b,c Mean
values ± standard error. Means not sharing a common superscript differ significantly (Tukey
p < 0.05).
2.2.2. ESEM
The microstructure and surface morphology of both adsorbent materials were scruti-
nized by ESEM (Figure 5). In general, the adsorbent derived from alfalfa leaves showed a
rough microstructure with large edges in the form of small sheets or ridges (Figure 5a,b).
This set of microstructures could play an important role during the adsorption of AFB1.
Shar et al. [29] suggest that functional groups and the heterogeneous microstructure on the
surface of the adsorbents contribute to the uptake of mycotoxins. On the other hand, in the
YCW, the microstructure of individual cells is clearly observed, with an assembly similar
to a raspberry [37], ellipsoid to oval in shape with a smooth surface and some invagina-
tions (Figure 5c,d). Hernández-Ramírez et al. [34], reported that the microstructure of the
Toxins 2023, 15, 604 7 of 18
YCW is characterized by its oval shape and smooth surface with aggregates of different
sizes. Unlike the alfalfa leaf-derived adsorbent, YCW has a notably less rough surface
and a less-exposed area, particularities that suggest that YCW would have a lower AFB1
adsorption capacity.
ff
  
(a) (b) 
  
(c) (d) 
Figure 5. ESEM micrographs of (a,b) the adsorbent material derived from alfalfa leaves and (c,d) the
yeast cell wall (YCW) at 2500× and 5000×, respectively.
2.2.3. XRF
With the micro X-ray fluorescence technique on ESEM, it was possible to perform
the micro-elemental analysis of both adsorbents. Figure 6 shows the XRF spectra of the
adsorbent derived from alfalfa leaves and the YCW. The elemental analysis of the adsorbent
prepared from alfalfa leaves showed a significant amount of carbon (49.44%), nitrogen
(5.48%), oxygen (43.92%), and traces of sodium (0.22%), magnesium (0.17%), aluminum
(0.27%), silicon (0.03%), phosphorus (0.04%), sulfur (0.07%), chlorine (0.07%), potassium
(0.18%), and calcium (0.11%). In this context, Zavala-Franco et al. [10] reported that the
main elements of the organic adsorbents they studied (banana peel, Pyracantha leaves,
and Aloe powder) were carbon and oxygen, which is consistent with our results. On the
other hand, in the XRF spectra of the YCW it can be seen that YCW has fewer elements;
however, there was a significant amount of carbon (45.32%), oxygen (38.02%), potassium
(7.87%), and traces of magnesium (1.32%), aluminum (0.50%), silicon (1.30%), phosphorus
(0.49%), sulfur (0.81%), and calcium (1.38%). In this regard, Chen et al. [38] showed that the
scanning electron micrograph of the Cinnamomum camphora leaf powder (CLP) adsorbent
was modified in terms of the change in intensity of some peaks after adsorption; these
changes in the intensity of the XRF peaks suggested that certain elements present in the
adsorbent were capable of effectively removing the pollutant [39]. Thus, the adsorbent
Toxins 2023, 15, 604 8 of 18
material derived from alfalfa leaves would have a better AFB1 adsorption capacity due to
the large amount of carbon and oxygen (up to 93.36%).
ff
tt
  
(a) (b) 
ff tt
ff
ff θ
θ
ff ff
θ θ
β
−
−
β
1 2 3 4
Na
Si
Cl
SP
Ca
Mg
K
Al
O
N
C
ou
nt
s
Energy (keV)
C
1 2 3 4
C
ou
nt
s
Energy (keV)
C
O
Mg
Al
Si
P S
K
Ca
Figure 6. Micro X-ray fluorescence spectra of (a) the adsorbent derived from alfalfa leaves and
(b) the yeast cell wall (YCW).
2.2.4. XRD
Figure 7 shows the X-ray diffraction pattern of the adsorbent material derived from
alfalfa leaves and the YCW. In the diffractogram of the adsorbent prepared from al-
falfa leaves (Figure 7a), a diffraction peak with a considerable intensity was observed at
2θ = 25.04◦. Wada et al. [40] associates this peak with the presence of crystalline cellulose.
Two other peaks of minor intensity at 2θ = 13.72◦ and 17.02◦ appeared, which were also
related to the presence of the cellulose type I crystalline structure [41] and semicrystalline
starch [10]. On the other hand, the diffractogram of the YCW (Figure 7b) showed three
diffraction peaks, the most intense at 24.88◦ 2θ and two less intense at 13.23◦ and 18.83◦
2θ. These peaks correspond to the polymeric crystalline structure of β-glucans [42,43].
The XRD results agree with what was found in the corresponding FTIR spectra of the
adsorbent materials. For instance, in the FTIR spectrum of the adsorbent derived from
alfalfa leaves, the band around 1408 cm−1 was associated with the presence of cellulose,
and in the spectrum of the YCW, the bands located at 887, 812, 670, 575, and 508 cm−1 were
associated with the presence of β-glucans.
 
(a) (b) 
ff tt
ζ
tt
ff
ff
ζ
ζ
ζ
ζ
ζ
ζ
− − −
ζ
tt ζ −
10 20 30 40 50 60 70
0
100
200
300
400
500
600
17.02°
13.72°
In
te
ns
ity
 (A
.U
)
2θ (degree)
25.04°
10 20 30 40 50 60 70
0
100
200
300
400
500
600
18.83°13.23°
In
te
ns
ity
 (A
.U
)
2θ (degree)
24.88°
Figure 7. X-ray diffraction patterns of (a) the adsorbent derived from alfalfa leaves and (b) the yeast
cell wall (YCW).
Toxins 2023, 15, 604 9 of 18
2.2.5. pHpzc and ζ-Potential
The pHpzc is a technique to characterize the surface charge of an adsorbent material.
It is well known that the adsorption capacity depends on the variation of the pH and the
degree to which the adsorbent and the adsorbate are protonated or deprotonated [44].
Figure 8a shows the pHpzc of the adsorbent materials. The pHpzc values of the adsorbent
prepared from alfalfa leaves and YCW were 6.51 and 2.45, respectively. Rosas-Castor
et al. [21] reported that the pHpzc of the alfalfa adsorbent was 6.9, and Hernández-Ramírez
et al. [34] reported that the pHpzc of the YCW was 3.09. According to these results, the
adsorbent derived from alfalfa leaves would have a better adsorption capacity at pH 2 and
5, that is, in the first two compartments of the GIT of birds, since the surface charge of the
adsorbent at these pH values remained positively charged, which favors the interaction
with the oxygen atoms of the AFB1 molecule. However, in the pH-dependent in vitro
experiment, there was no statistically significant difference in the adsorption of AFB1 at the
three pH values evaluated; thus, it can be hypothesized that at pH 7 electrostatic interaction
is not the main adsorption mechanism. These results agree with Adunphatcharaphon
et al. [26], who evaluated the effect of pH on AFB1 adsorption using durian peel.
  
(a) (b) 
ff
ff
ff ff
ffi
tt
2 4 6 8 10 12
-4
-3
-2
-1
0
1
2
3
4
5
6
7
Δ
pH
pH
 Alfalfa
 Yeast cell wall
2 4 6 8 10 12
-22
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
2
4
Ze
ta
 P
ot
en
tia
l (
m
V)
pH
 Alfalfa
 Yeast cell wall
Figure 8. Point of zero charge (a) and Zeta potential (b) of the adsorbent materials.
On the other hand, ζ-potential is a technique that allows knowing the electric field orig-
inated by the surface charges of a certain material [45,46]. In this research, ζ-potential was
determined in a pH range from 2 to 11 (Figure 8b). In general, the ζ-potential of the two
tested adsorbents was more negative as the pH increased. Electronegative values of ζ-potential
indicate a large accumulation of positive charges near the particle surface. On the contrary,
electropositive ζ-potential values allow the accumulation of negative charges near the particle
surface [46]. Thus, the ζ-potential of the YCW was negative in the entire pH scale that involves
the GIT: pH 2 (−0.81 mV), pH 5 (−5.77 mV), and pH 7 (−5.95 mV). On the contrary, the
ζ-potential of the adsorbent derived from alfalfa leaves was positive at pH 2 (+2.63 mV), and it
was significantly more negative than the YCW in two of the three GIT compartments (pH 5 and
pH 7), attaining ζ-potential values of −14.55 mV and −14.91 mV, respectively. Therefore, it is
proposed that there is a significant contribution to the AFB1 adsorption when using the alfalfa
leaf-derived adsorbent compared to the reference material at acidic pH values (pH 2), due to
electrostatic interactions between the adsorbent and the mycotoxin [47].
2.2.6. Determination of Chlorophylls and Carotenoids
Spectral Reflectance Measurements
To evaluate the presence of pigments in the adsorbent materials, the UV-Vis diffuse
reflectance spectroscopy technique was utilized [12]. Figure 9 shows the diffuse reflectance
spectra of the adsorbent derived from alfalfa leaves and the reference material (YCW). The
Toxins 2023, 15, 604 10 of 18
adsorbent prepared from alfalfa leaves showed two representative bands (678 nm and
650 nm), which correspond to Chl a and Chl b, respectively. Moreover, the absorbance
of anthocyanins is associated with the maxima at 550 nm [27]. Finally, in the range from
430 to 530 nm, the presence of carotenoids was observed [48]. These results agree with
Merzlyak et al. [49], who studied the diffuse reflectance spectra of five different apple peels.
The authors reported the presence of chlorophylls a and b, carotenoids, and anthocyanins.
On the contrary, in the YCW, no representative bands were found (Figure 9). Nava-
Ramirez et al. [31] conducted a study to test the efficacy of three adsorbents with a high
chlorophyll content (lettuce, pyracantha, and horsetail) for AFB1 adsorption; the researchers
demonstrated that the more chlorophylls an adsorbent has, the greater the adsorption
capacity. The authors concluded that chlorophylls were able to form strong non-covalent
complexes with the AFB1 molecule. In this research, the adsorbent derived from alfalfa
leaves contained large amounts of chlorophylls; therefore, it had higher possibilities to
form chlorophyll–AFB1 complexes. This ability to form chlorophyll–AFB1 complexes could
explain the great adsorption potential of the adsorbent derived from alfalfa leaves.
 
ff
400 500 600 700 800
0.0
0.2
0.4
0.6
0.8
1.0
1.2
C
ar
ot
en
oi
ds
A
nt
ho
cy
an
in
s
C
hl
 b
Ab
so
rb
an
ce
 (A
.U
.)
Wavelength (nm)
 Alfalfa
 Yeast cell wall
C
hl
 a
Figure 9. Diffuse reflectance UV-Vis spectra of the adsorbent materials.
Quantitative Determination of Chlorophylls and Carotenoids
To perform the quantification of pigments in both adsorbent materials, Chl a, Chl b, and
total carotenoids were extracted with 96% ethanol and subsequently the absorbance of each
pigment was determined spectrophotometrically. The spectrum of the adsorbent derived from
alfalfa leaves (Figure S1) shows a narrow maximum band in the blue region (near 432 nm)
and another band in the red region (near 665 nm). These bands correspond to the presence of
Chl a [27,33] [27,33]. Carotenoids have a broad absorption with three shoulders within the
blue region between 400 and 500 nm [50]. To know the real content of the main photosynthetic
pigments in the alfalfa adsorbent, the specific absorption coefficients of Lichtenthaler and
Wellburn [51] were used (Table 2). It was observed that the total chlorophyll content of the
adsorbent prepared from alfalfa leaves (2759.1 ± 180.2 µg/g dry weight) was significantly
higher than the total carotenoid content (16.3 ± 94.5 µg/g dry weight). The chlorophyll
content in alfalfa agrees with that reported by Dziwulska-Hunek et al. [52]. Figure S1 also
shows the UV-Vis spectrum of the YCW ethanolic extract; it is clearly observed that this
adsorbent does not contain pigments.
Toxins 2023, 15, 604 11 of 18
Table 2. Chlorophylls and total carotenoid contents of the adsorbent materials.
Photosynthetic Pigment
Content (µg/g Dry Weight)
Alfalfa YCW
Chlorophyll a 1251.2.1 ± 84.4 ND
Chlorophyll b 1508.0 ± 132.8 ND
Total chlorophyll (a + b) 2759.1 ± 180.2 ND
Total carotenoid (x + c) 16.3 ± 94.5 ND
x + c = xantophyll + carotenes. ND = not detected.
Furthermore, Figure 10 shows the fluorescence spectrum of the chlorophylls of the
adsorbent materials. The spectrum of the adsorbent prepared from alfalfa leaves shows
two fluorescence maximums, one at 690 nm and the other at 735 nm, which correspond to
the red and far-red chlorophyll fluorescence, respectively [27,53]. The fluorescence ratio of
these two bands F690/F735 was 8.3. It has been reported that this ratio is a useful tool to
detect variations in chlorophyll content in plants and this ratio decreases with increasing
the chlorophyll content when re-absorption processes are excluded [27]. Buschmann [54]
reported that the F690/F735 ratio of a diluted leaf extract was 5.7 and this ratio decreased to
0.37 with increases in the chlorophyll concentration (from 3 to 159 µg/mL). In this research,
in the fluorescence spectrum of the YCW ethanolic extract, it is clearly observed that this
adsorbent material does not contain any pigments.
π π
tt
tt
π π
ff
ffi
 
 
 
600 650 700 750 800
0
100
200
300
400
500
600
700
800
Fl
uo
re
sc
en
ce
 in
te
ns
ity
 (A
.U
.)
Wavelength (nm)
 Alfalfa
 YCW
735 nm
690 nm
Figure 10. Chlorophyll fluorescence spectra of the adsorbent materials.
3. The Proposed Adsorption Mechanism
Adsorbent materials are capable of removing AFB1 through a combination of chemi-
cal (redox reactions, complex formation, covalent bonds, and proton displacement) and
physical (hydrophobic interactions, π–π stacking, dipole–dipole interactions, hydrogen
bonding, and Van der Waals forces) mechanisms [55]. These mechanisms can be divided
into electrostatic (ionic attractions) and non-electrostatic (dipole–dipole, hydrogen bond-
ing, and hydrophobic) interactions [29]. Electrostatic interactions depend on the pH of
the solution; therefore, in the present study, ionic attractions play a minor role during
AFB1 adsorption. On the other hand, due to the significant presence of hydroxyl, amino,
and amide groups, the adsorbent material derived from alfalfa leaves would construct
numerous hydrogen bonding networks with the oxygen atoms of the ether, carbonyl, and
methoxy groups of the AFB1 molecule. Consequently, we propose that this interaction
is one of the main mechanisms for AFB1 adsorption. In addition, the considerable pres-
ence of hydrophobic groups such as methyl and aromatics in the adsorbent derived from
alfalfa leaves was favorable for the formation of hydrophobic, dipole–dipole, and π–π
Toxins 2023, 15, 604 12 of 18
stacking interactions [13,27]. Finally, it has been previously described—by our research
group—that chlorophyll, which is a highly hydrophobic molecule, can form strong non-
covalent complexes with the AFB1 molecule, regardless of pH [31]. Therefore, the for-
mation of these complexes also contributed to the AFB1 adsorption. The set of these
electrostatic and non-electrostatic interactions makes the adsorbent derived from alfalfa
leaves an effective material for the adsorption of AFB1 from the contaminated feed destined
for poultry.
4. Conclusions
Taken together, these findings suggest that the use of an adsorbent material derived
from alfalfa leaves is a viable alternative for removing AFB1 in in vitro digestion models
that closely mimic the complex physiological conditions of birds. Both in vitro trials
demonstrated that the plant-based adsorbent was efficient for the adsorption of AFB1
due to the combination of electrostatic and non-electrostatic interactions. Therefore, this
material can be used at a low inclusion level (0.5% w/w), to successfully remove the
AFB1 present in poultry feed. However, since in vitro models do not perfectly mimic
the GIT conditions, in vivo studies are also required to help determine the efficacy of the
alfalfa adsorbent in reducing the toxic effects of AFB1. Our group of researchers is already
conducting these relevant studies. The results will be published elsewhere.
5. Materials and Methods
5.1. Chemicals and Reagents
The AFB1 standard (CAS number: 1162-65-8), dimethyl sulfoxide (≥99.9% purity, CAS
number 67-68-5), HPLC grade methanol (CAS number 67-56-1), 96% ethanol (CAS number
64-17-5), sodium hydroxide (NaOH; ≥97% purity; CAS number 1310-73-2), hydrochloric
acid (HCl; ~37% purity; CAS number 7647-01-0), and sodium hypochlorite solution (CAS
number 7681-52-9) were acquired from Merck KGaA (Darmstadt, Germany).
5.2. Adsorbent Materials
The in vitro experiments were carried out with alfalfa leaves (Medicago sativa L.),
collected in the Botanic Garden of the Superior Studies Faculty at Cuautitlan (National
Autonomous University of Mexico). A commercial premium yeast cell wall (YCW) from
Saccharomyces cerevisiae (SafMannan, Phileo Lesaffre Animal Care, Lesaffre Iberica S.A.,
Valladolid, Spain) was used as a reference material. Briefly, alfalfa leaves were separated
manually and thoroughly washed with distilled water to remove surface-adhered dirt
particles. Subsequently, the fresh leaves were dried for 24 h at a constant temperature
(40 ◦C) in an oven (Binder model RE-115, Tuttlingen, Germany). The dried leaves were
ground in a C-11-1 type electric plate mill (Glen Mills Inc., Clifton, NJ, USA). Since adsorp-
tion is affected by varying the particle size, the ground material was sieved to obtain a
size within the optimal range for AFB1 adsorption, attaining an average size of <250 µm
(60 mesh sieve) as recommended by Zavala-Franco et al. [10]. Finally, the freshly prepared
material was stored in a vacuum-sealed plastic container and kept in a desiccator until
use. In the in vitro experiments, an inclusion level of 0.5% (w/w) was utilized, which is in
accordance with the safe limit that the Panel on Additives and Products or Substances used
in Animal Feed (FEEDAP) considers for a mycotoxin binder montmorillonite [11].
5.3. In Vitro Adsorption Studies
5.3.1. Preparation of the Aflatoxin B1 (AFB1) Solution and the pH-Dependent Model
A stock solution of AFB1 (100 µg/mL, equivalent to 0.32 mM) was prepared by
dissolving the toxin in dimethyl sulfoxide (DMSO). The stock solution was stored in
the dark at 4 ◦C until further use. Then, the stock solution was diluted in deionized
water adjusted at three pH values (2, 5, and 7) until reaching a final concentration of
250 ng AFB1/mL.
Toxins 2023, 15, 604 13 of 18
To assess the efficacy of the adsorbent in removing AFB1, a single in vitro model was
considered. Samples of 25 mg (0.5% w/w) of the adsorbent materials were accurately
weighed and dispersed in glass vials with 5 mL of the solutions containing the AFB1. The
flasks were incubated in an agitated water bath (Bellco Glass Inc. Vineland, NJ, USA)
and carefully homogenized at 120 rpm at a temperature of 40 ◦C for 2 h. Subsequently,
the adsorbent was separated by centrifugation at 7000× g (5810 R centrifuge, Eppendorf,
Hamburg, Germany) for 7 min, and the supernatant was filtered through a PTFE membrane
syringe filter (pore size 0.22 µm) and subjected to ultra-performance liquid chromatography
with fluorescence detection (UPLC-FLR). The pH was determined using a glass electrode
(Conductronic PC-45, Puebla, Mexico). All determinations were performed in quintuplicate.
Control samples (without the addition of adsorbents) were also included in the experiment
to confirm the stability of the AFB1 molecule in the different pH media under the same
incubation conditions.
5.3.2. Preparation of the AFB1-Contaminated Diet and the Avian Intestinal Model
A commercial maize-soybean meal poultry diet (Nutricion Tecnica Animal SA de
CV, Queretaro, Mexico) containing 26% protein (12.64 MJ/kg metabolizable energy) was
contaminated at a content of 250 µg AFB1/kg. Levels of AFB1, total fumonisins, and
deoxynivalenol were determined in the commercial feed using VICAM’s fluorometric
tests based on monoclonal antibody-based affinity columns (VICAM Science Technology,
Watertown, MA, USA). To check the homogeneity of the aflatoxin-contaminated feed,
five random samples were taken, and the presence of AFB1 was confirmed using the
991.31 AOAC procedure [56].
An avian intestinal model was used to evaluate the adsorption capacity of the adsor-
bent materials using the procedure reported by Hernandez-Patlán et al. [57] with minimal
modifications. The test was carried out with one reference material (YCW) and one treat-
ment (the adsorbent prepared from alfalfa leaves). The experiment was conducted at
40 ◦C to emulate the poultry body temperature with constant agitation (19 rpm). In the first
stage, 5 g of the AFB1-contaminated feed and 0.5% (w/w) of the adsorbent material was
mixed and poured into polypropylene centrifuge tubes (50 mL capacity). To emulate the
crop environment, 10 mL of 0.03 M HCl were added, and tubes were incubated for 30 min.
The pH value was around 5. After this period, 2.5 mL of 1.5 M HCl and 3000 U of pepsin
(Merck KGaA, Darmstadt, Germany) per gram of feed was added to each tube to emu-
late the proventriculus environment, reaching a pH of 2. Tubes were incubated again for
45 min. Finally, to emulate the intestinal section, 6.84 mg of 8× pancreatin (Merck KGaA,
Darmstadt, Germany) in 6.5 mL of 1.0 M NaHCO3 was added to the tubes and incubated
for another 120 min. The entire avian intestinal model simulation took 195 min. At the
end of the assay, the tubes were centrifuged at 7000× g for 10 min, and the residual AFB1
concentration remaining in the supernatant was quantified by the UPLC-FLR technique.
All determinations were also performed in quintuplicate. Control samples (without the
addition of adsorbent materials) were also included to know the real concentration of AFB1
per tube under the simulated conditions of the gastrointestinal tract of birds.
The percentage of AFB1 adsorbed was computed using the following
mathematical expression:
Adsorption (%) =
(Ci − Cs)
Ci
× 100 (1)
where Ci is the concentration of AFB1 in the control samples (ng/mL), and Cs is the
concentration of AFB1 in the supernatant (ng/mL).
5.3.3. Analysis of Aflatoxin B1 (AFB1)
To quantify the AFB1 concentration in the supernatants, immunoaffinity columns
based on monoclonal antibodies (Afla-B, VICAM Science Technology, Watertown, MA,
USA) were used as a clean-up protocol. Subsequently, the purified toxin was analyzed
Toxins 2023, 15, 604 14 of 18
by UPLC-FLR using a Waters ACQUITY H-class System. Briefly, the methanolic extracts
collected from the immunoaffinity columns (1 µL) were injected and eluted with a mobile
phase of water:methanol:acetonitrile (64:18:18) at a flow rate of 700 µL/min. The AFB1 was
detected using excitation and emission wavelengths of 365 nm and 435 nm, respectively.
The AFB1 concentration was estimated using a reference standard (AFB1, Merck KGaA,
Darmstadt, Germany) with a calibration curve. The detection limit of AFB1 was found to
be 0.002 µg/L.
5.4. Characterization
5.4.1. Fourier Transform Infrared Spectroscopy with Attenuated Total
Reflection (FTIR-ATR)
Infrared spectra were collected using a Frontier SP8000 Fourier transform infrared
spectrophotometer (Perkin Elmer, Waltham, MA, USA) equipped with an attenuated total
reflection (ATR) attachment (DuraSamplIR II, Smiths Detection, Warrington, UK). The
adsorbents were scanned in the 4000–400 cm−1 region. The bond indexes (BIs) of the
principal functional groups were calculated using the following mathematical expressions:
BIOH =
BA3281
∑ BA
(2)
BI(CH2)n =
BA2916 + BA2850
∑ BA
(3)
BICOOR =
BA1613
∑ BA
(4)
BIC=C =
BA1406
∑ BA
(5)
BIC−O =
BA1031
∑ BA
(6)
where BAx is the band area around the corresponding wavenumber (cm−1); and ΣBA is
the total area of all bands in the corresponding FTIR spectrum.
5.4.2. Environmental Scanning Electron Microscopy (ESEM)
The microstructure and morphology of the adsorbents were characterized by environ-
mental scanning electron microscopy (Philips-XL30 ESEM, Eindhoven, The Netherlands)
with an accelerating voltage of 3, 5, and 20 kV. Samples were coated with a thin layer
of gold using a sputter coater (Denton Vacuum Inc., Desk V HP, Moorestown, NJ, USA)
operated at 7 mA for 3 min. Microscopy analysis (2500× and 5000×) was performed in a
secondary electron imaging (SEI) mode.
5.4.3. X-ray Fluorescence Spectroscopy (XRF)
The multi-elemental analysis was conducted in triplicate, using a high-performance
XTrace microspot X-ray source, and the photon-induced micro-X-ray fluorescence spectrum
was measured with the XFlash® 6/10 silicon drift detector (Bruker Nano GmbH, Berlin,
Germany). The technique was carried out using an environmental scanning electron
microscope equipped with X-ray fluorescence spectroscopy (Phillips XL30/40 XRF-ESEM,
Eindhoven, The Netherlands).
5.4.4. X-ray Diffraction (XRD)
The X-ray diffraction measurements were conducted on a 2100-Rigaku diffractometer
(Rigaku Co., Tokyo, Japan). The diffraction data were recorded for 2θ between 5◦ and 70◦
with a resolution of 0.02◦.
Toxins 2023, 15, 604 15 of 18
5.4.5. Point of Zero Charge (pHpzc) and Zeta Potential (ζ-Potential)
The point of zero charge (pHpzc) was determined following the procedure described
by Zavala-Franco et al. [10]. In brief, samples of each of the adsorbent materials (25 mg)
were weighed in five flasks containing deionized water adjusted at different pH values
(pHi=2, 5, 7, 9, and 11). Subsequently, samples were shaken at 200 rpm at room temperature
for 195 min. Thereafter, the final pH (pHf) of the supernatant was recorded using a glass
electrode. A plot of pHpzc was constructed as follows: ∆pH (pHi–pHf) against pHi. On the
other hand, Zeta potential (ζ-potential) measurements were performed using the ZetaSizer
Pro (Malvern Instruments, Worcestershire, UK) following the recommendations of Ramales-
Valderrama [11]. All determinations were conducted at room temperature in quintuplicate.
5.4.6. Chlorophyll and Carotenoid Quantification
A Lambda 365 UV-Vis-diffuse reflectance spectrophotometer (Perkin Elmer, Waltham,
MA, USA) equipped with a 100-mm integrating sphere was used. The optical absorp-
tion spectra of powders were collected in the range of 400–800 nm. The pigments such
as chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoids (xanthophylls +
carotenes) were extracted with 96% ethanol and characterized using a Cary 8454 UV-
Vis Diode Array System spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).
The absorption coefficients reported by Lichtenthaler and Wellburn [51] were utilized for
pigment quantification.
5.5. Experimental Design and Statistical Analysis
The experiments were conducted as a completely randomized design, and data were
analyzed by means of a one-way analysis of variance (ANOVA). The Tukey test was used
to compare the means from the ANOVA. The threshold for significance level was set at
α = 0.05. The OriginPro v8 software was used.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/toxins15100604/s1, Figure S1: UV-Vis spectrum of the main
pigments contained in the adsorbent materials.
Author Contributions: Conceptualization, A.M.-A. and C.L.-C.; methodology, M.d.J.N.-R., D.H.-
P. and B.S.-C.; formal analysis, J.d.D.F.-C., A.V.-D. and M.d.J.N.-R.; resources, A.M.-A. and G.T-I.;
data curation, M.d.J.N.-R., D.H.-P. and B.S.-C.; writing—original draft preparation, M.d.J.N.-R. and
A.M.-A.; writing—review and editing, A.M.-A., C.L.-C. and G.T.-I.; supervision, A.M.-A., A.V.-D.,
C.L.-C., J.d.D.F.-C. and G.T-I.; project administration, A.M.-A. and A.V.-D. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was supported by UNAM-PAPIIT grant number IA101523 and by funds pro-
vided by USDA-NIFA, Sustainable Agriculture Systems, grant number 2019-69012-29905.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets used and or analyzed during the current study are
available from the corresponding author upon reasonable request.
Acknowledgments: The authors wish to thank the expert technical assistance of José Juan Vélez Med-
ina, José Eleazar Urbina Álvarez, and Martín Adelaido Hernández Landaverde from CINVESTAV-
IPN Unidad Queretaro in ESEM, EDXRS, and XRD characterization. María de Jesus Nava Ramírez
acknowledges CONAHCYT for the scholarship CVU. 866355.
Conflicts of Interest: The authors declare no conflict of interest.
Toxins 2023, 15, 604 16 of 18
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Vol.:(0123456789)
Mycotoxin Research (2024) 40:269–277 
https://doi.org/10.1007/s12550-024-00527-4
ORIGINAL ARTICLE
Efficacy of powdered alfalfa leaves to ameliorate the toxic effects 
of aflatoxin  B1 in turkey poults
M. J. Nava‑Ramírez1 · J. A. Maguey‑González2 · S. Gómez‑Rosales3 · J. O. Hernández‑Ramírez1 · J. D. Latorre2 · 
Xiangwei Du4 · C. López‑Coello5 · B. M. Hargis2 · G. Téllez‑Isaías2 · A. Vázquez‑Durán1 · A. Méndez‑Albores1 
Received: 25 October 2023 / Revised: 19 February 2024 / Accepted: 22 February 2024 / Published online: 29 February 2024 
© The Author(s) 2024
Abstract
This experiment was conducted to determine the effect of an adsorbent material based on powdered alfalfa leaves added in the 
aflatoxin  B1  (AFB1)-contaminated diet of turkey poults on production parameters, blood cell count, serum biochemistry, liver 
enzymes, and liver histology. For this purpose, three hundred and fifty female Nicholas-700 poults were randomly assigned 
into five treatments: (1) Control,  AFB1-free diet; (2) AF, diet contaminated with 250 ng  AFB1/g; (3) Alfalfa,  AFB1-free 
diet + 0.5% (w/w) adsorbent; (4) AF+alfalfa, diet contaminated with 250 ng  AFB1/g + 0.5% (w/w) adsorbent, and (5) AF+ 
yeast cell wall (YCW), diet contaminated with 250 ng  AFB1/g + 0.5% (w/w) of yeast cell wall (a commercial mycotoxin 
binder used as reference material). The in vivo efficacy of powdered alfalfa leaves was assessed during a 28-day period. In 
general, the addition of powdered alfalfa leaves in the  AFB1-free diet gave the best performance results (body weight, body 
weight gain, and feed intake) and improved the values of total protein, glucose, calcium, creatinine, and blood urea nitrogen. 
Moreover, the addition of powdered alfalfa leaves in the  AFB1-contaminated diet enhanced body weight and body weight 
gain and significantly reduced the feed intake, compared to the AF and AF+YCW groups. Additionally, significant altera-
tions in serum parameters were observed in poults intoxicated with the  AFB1, compared to the Control group. Furthermore, 
typical histopathological lesions were observed in the liver of the AF group, which were significantly ameliorated with the 
addition of powdered alfalfa leaves. Conclusively, these results pointed out that low inclusion of powdered alfalfa leaves in 
the contaminated feed counteracted the adverse effects of  AFB1 in turkey poults.
Keywords Turkey poults · Aflatoxin  B1 · Powdered alfalfa leaves · Adsorption · Performance · Histology
Introduction
Aflatoxins are mycotoxins composed of a pentacyclic struc-
ture with a difuran and coumarin skeleton and are mainly 
produced by fungal species of the genus Aspergillus (Lala 
et al. 2016). Aflatoxin  B1  (AFB1) is ubiquitous in rations 
intended for poultry consumption, and this molecule pos-
sesses the most powerful carcinogenic, teratogenic, muta-
genic, and immunosuppressive potential (Rawal et al. 2010). 
Turkeys are found among the most susceptible species to 
 AFB1 due to their deficient detoxification mechanism in the 
liver (Reed et al. 2019). Consumption of feed contaminated 
with considerable amounts of  AFB1 causes various adverse 
effects in poultry such as alteration in feed consumption, 
weight gain, increased morbidity and mortality, hematologi-
cal and biochemical changes, and in some cases reduction in 
the relative weight of immune organs, as well as important 
macroscopic and microscopic changes in the liver (Grozeva 
 * A. Méndez-Albores 
 albores@unam.mx
1 Unidad de Investigación Multidisciplinaria L14 (Alimentos, 
Micotoxinas, y Micotoxicosis), Facultad de Estudios 
Superiores Cuautitlán, Universidad Nacional Autónoma de 
México (UNAM), Cuautitlán Izcalli 54714, Mexico
2 Department of Poultry Science, University of Arkansas, 
Fayetteville, AR 72701, USA
3 Centro Nacional de Investigación Disciplinaria en Fisiología 
y Mejoramiento Animal (CENID-INIFAP), Km 1 Carretera a 
Colon Ajuchitlán, Querétaro 76280, Mexico
4 Veterinary Medical Diagnostic Laboratory, College 
of Veterinary Medicine, University of Missouri, Columbia, 
MO 65211, USA
5 Departamento de Medicina y Zootecnia de Aves, Facultad 
de Medicina Veterinaria y Zootecnia, Universidad Nacional 
Autónoma de México (UNAM), Ciudad de Mexico 04510, 
Mexico
270 Mycotoxin Research (2024) 40:269–277
et al. 2020). Currently, the incidence of  AFB1 contamina-
tion in grains destined for feed production has had great-
est variability due to global climate change; consequently, 
the development of environmentally friendly strategies is 
required to guarantee the safety of certain feed ingredients 
(Khodaei et al. 2021). In this context, the most used strat-
egy for the physical control of aflatoxins in feedstuffs is the 
addition of inorganic adsorbent materials such as zeolites, 
aluminosilicates, hydrated sodium calcium aluminosilicate 
(HSCAS), clays, among others. In recent years, plant-based 
adsorbents have turned out to be promising compared to 
inorganic adsorbents since they have been shown to be effec-
tive and more cost-effective (Vila-Donat et al. 2018).
Several in vitro studies have been carried out using agri-
cultural wastes as adsorbent materials for  AFB1 removal. In 
these studies, the main effects of several variables such as 
pH, temperature, time, and dose (adsorbent/adsorbate) have 
been evaluated. Very recently, our research group carried out 
an investigation to determine the effectiveness of an adsor-
bent made from powdered alfalfa leaves for the removal of 
 AFB1 using two in vitro models (Nava-Ramírez et al. 2023). 
When using a pH-dependent model, adsorption values above 
98% were obtained and when an avian intestinal model was 
utilized, a considerable reduction in the  AFB1 uptake by 
the powdered alfalfa leaves was observed (88.8%). How-
ever, until now, few in vivo studies have been carried out 
for the evaluation of the efficacy of plant-based materials 
as  AFB1 adsorbents (Gambacorta et al. 2016; Perali et al. 
2020; Taranu et al. 2020).
It has been reported that the addition of alfalfa to the diet of 
poultry significantly improved body weight, body weight gain, 
growth performance, and the reproductive capacity. Addition-
ally, a positive effect has also been observed in some serum 
biochemical parameters, and the height of the villi and depth 
of the duodenal crypt of the intestine (Suwignyo and Sasongko 
2019). To the best of our knowledge, the in vivo efficacy of 
alfalfa as an  AFB1 adsorbent material has not been reported; 
consequently, the aim of the present research was to evaluate 
the effect of powdered alfalfa leaves to ameliorate the toxic 
effects of  AFB1 in turkey poults.
Materials and methods
Preparation of the adsorbent material
Alfalfa (Medicago sativa L.) leaves collected from the 
botanic garden of the National Autonomous University 
of Mexico-Superior Studies Faculty at Cuautitlan were 
washed with distilled water and dried in an oven (Binder 
model RE-115, Tuttlingen, Germany) at 40 °C for 48 h. 
The dried leaves were finely ground in an electric plate 
mill (Glen Mills Inc., Clifton, NJ, USA) and subsequently 
passed through a 60 mesh to obtain a particle size distribu-
tion of < 250 µm. Finally, the powdered alfalfa leaves were 
deposited in a plastic container and placed in a desiccator 
over silica pellets. A commercial aflatoxin binder based 
on yeast cell wall (YCW) from Saccharomyces cerevisiae 
(SafMannan, Phileo Lesaffre Animal Care, Lesaffre Iberica 
S.A., Valladolid, Spain) was used as a reference material.
Aflatoxin production
Aflatoxins were produced on rice according to the recom-
mendations of Shotwell et al. (1966). A spore suspension 
of the Aspergillus flavus strain NRRL 2999 was utilized to 
inoculate the solid substrate in 250-mL Erlenmeyer flasks 
(50 g of rice + 25 mL water + 0.5 mL of the conidia suspen-
sion). Flasks were incubated in a New Brunswick incubator 
at 28 °C for 5 days under agitation (188 rpm) at the Vet-
erinary Medical Diagnostic Laboratory of the University 
of Missouri, Columbia, USA. At the end of the incubation 
period, the aflatoxin-contaminated rice was steam-sterilized 
(121 °C, 106 kPa, 15 min), dried, finely ground, and the 
aflatoxin content was estimated by means of liquid chro-
matography with fluorescence detection according to the 
recommendations of Göbel and Lusky (2004).
Preparation of the aflatoxin‑contaminated diet 
and mycotoxin analyses
To assure a proper distribution of the aflatoxins, the highly con-
taminated rice was previously mixed in the commercial turkey 
poult diet (Nutricion Tecnica Animal SA de CV, Queretaro, 
Mexico) to a content of 25,000 ng  AFB1 per gram of feed. 
This stock was subsequently used to contaminate the rest of the 
commercial feed, using 10 g of the stock per kg of feed. The 
composition of the diet was similar to previously reported data 
for turkeys (Maguey-González et al. 2023). Batches of 30 kg 
were artificially contaminated to give a final content of 250 ng 
 AFB1/g feed. Before the contamination process, the commer-
cial turkey poult diet was analyzed for total aflatoxins  (AFB1, 
 AFB2,  AFG1, and  AFG2), total fumonisins  (FB1,  FB2, and  FB3), 
and deoxynivalenol (DON) following the recommendations of 
VICAM, Science Technology, Watertown, MA, USA (https:// 
www. vicam. com/ categ ory/ aflat oxin- testi ng- solut ions, https:// 
www. vicam. com/ categ ory/ fumon isin- testi ng- solut ions, and 
https:// www. vicam. com/ toxins/ deoxy nival enol, accessed on 
08 February 2024). The adsorbent materials (powdered alfalfa 
leaves and YCW) were also mixed in the respective diet at an 
inclusion level of 0.5% (w/w). Finally, five aflatoxin-contam-
inated feed samples were taken at random, and the content of 
 AFB1 was estimated according to the 991.31 AOAC methodol-
ogy (Horwitz 2010).
271Mycotoxin Research (2024) 40:269–277 
Experimental design of in vivo experiment
All procedures on experimental animals were in accord-
ance with and approved by the Institutional Animal Care 
and Use Committee (IACUC) at the University of Arkan-
sas under protocol number 22020. Three hundred and fifty 
one-day-old female Nicholas-700 turkey poults (Aviagen 
Inc., AR, USA) were randomly distributed in five pens with 
seven repetitions each (n = 70 per treatment) as follows: (1) 
Control,  AFB1-free diet; (2) AF, diet contaminated with 
250 ng  AFB1/g; (3) Alfalfa,  AFB1-free diet + 0.5% (w/w) 
adsorbent; (4) AF + alfalfa, diet contaminated with 250 ng 
 AFB1/g + 0.5% (w/w) adsorbent, and (5) AF+YCW, diet 
contaminated with 250 ng  AFB1/g + 0.5% (w/w) of YCW 
(a commercial mycotoxin binder used as reference mate-
rial). Poults were maintained for 28 days with free access to 
feed and water. The temperature and lighting programs were 
followed according to the recommendations of the supplier 
(Aviagen 2015).
Collection of samples and measurements
Poults and feed were weighed on a weekly basis to calculate 
the body weight (BW), body weight gain (BWG), feed conver-
sion ratio (FCR), and feed intake (FI). Mortality was recorded 
throughout the experiment. At the end of the trial, 21 turkeys 
were randomly selected from each treatment (three poults per 
replicate) and samples of whole blood were taken and serum 
prepared. Serum analysis was performed with a spectrophotom-
eter using commercially available kits (BioSystems, Barcelona, 
Spain) to determine total protein (TP), glucose (Glu), calcium 
(Ca), uric acid (UA), creatinine (CRE), blood urea nitrogen 
(BUN), and the enzymes alanine aminotransferase (ALT), alka-
line phosphatase (ALP), aspartate aminotransferase (AST), cre-
atine kinase (CK), and glutamate dehydrogenase (GLDH). The 
determination of hematocrit (Hct), and cell count of leukocytes 
(WBCs), lymphocytes (Ls), basophils (Ba), monocytes (Mn), 
and heterophils (H) was performed using an automated hema-
tology analyzer (Cell-Dyn 1700; Abbott Diagnostics, Abbott 
Park, IL, USA), following the recommendations of Maguey-
González et al. (2023). Finally, the bled poults were sacrificed 
by inhalation of 80% carbon dioxide, 5% oxygen, and 15% 
nitrogen (Coenen et al. 2000). The liver, spleen, and bursa of 
Fabricius were removed, rinsed with cold saline, and weighed. 
For the histological study, liver samples were selected from the 
left hepatic lobe and fixed in 10% neutral buffered formalin, 
embedded in paraffin, cut into 5-μm thick sections, and stained 
with the hematoxylin and eosin (H&E) technique. The slides 
were examined at 40× magnification. The classification scheme 
of the semiquantitative evaluation of the liver lesions was as 
follows: severity grade 0 (lesion not present or within normal 
levels), grade 1 (mild lesion), grade 2 (moderate lesion), and 
grade 3 (severe lesion).
Statistical analysis
Data on productive parameters, relative organ weight, blood 
count cells, biochemical, and enzymatic analysis were sub-
jected to analysis of variance (ANOVA) using the General 
Linear Model (GLM) procedure in the Statistical Analysis 
System software version 8.0 (SAS Institute Inc. Cary, NC, 
USA). Means were separated by the Tukey procedure. The 
Kruskal-Wallis non-parametric test was performed to assess 
the histological analysis. A value of p < 0.05 was considered 
to reject null hypothesis.
Results and discussion
Analysis of dietary aflatoxins
In general, the uncontaminated commercial feed samples 
analyzed contained slightly higher than the lowest detect-
able content of the total aflatoxins, total fumonisins, and 
deoxynivalenol. The contents of these fungal toxins in the 
commercial diet were 1.7 ng/g, 0.02 mg/kg, and 0.09 mg/kg, 
respectively. Thus, the presence of these mycotoxins in the 
feed was considered to be negligible (Méndez-Albores et al. 
2005). Moreover, the artificially aflatoxin-contaminated tur-
key diet contained 250 ± 14 ng  AFB1/g, analyzed by means 
of the 991.31 AOAC methodology.
Production parameters
The production parameters of the turkey poults from day 
1 to day 28 are shown in Table 1. At the beginning of the 
experiment (day old poults), no statistically significant dif-
ferences were found between the five treatments in terms 
of BW. However, in the last week of the trial (28 day old), 
it was observed that turkeys that received the diet contami-
nated with  AFB1 had a significant reduction in BW, show-
ing a deviation of −12.94%, compared to the Control group. 
Oyegunwa et al. (2021) reported that offering an experi-
mental diet contaminated with 200 ng  AFB1/g feed during a 
28-day period produced a significant decrease on turkey BW 
(up to 33% compared to the control group). Other authors 
also reported a significant reduction in BW in broiler chick-
ens fed a diet contaminated with 200 ng  AFB1/g (Tessari 
et al. 2006). On the contrary, the marked reduction in BW 
caused by the  AFB1 consumption, improved significantly 
(p < 0.0001) with the use of powdered alfalfa leaves; conse-
quently, poults of the AF+alfalfa group presented a devia-
tion of − 4.4% compared to the Control group. Furthermore, 
with the use of YCW, a significant reduction in the BW was 
also observed in the AF+YCW group showing a deviation 
of −10.2% compared to the Control group. These results 
agree with the findings reported by Hernández-Ramírez 
272 Mycotoxin Research (2024) 40:269–277
et al. (2021), who reported that the addition of YCW (0.05%) 
to a diet contaminated with  AFB1 (500 ng/g) did not allevi-
ate the negative effects caused by the mycotoxin in broiler 
chickens. Interestingly, the treatment that was provided with 
the powdered alfalfa leaves (Alfalfa) had a significantly 
higher BW, up to 528.21 g/poult (7.41 deviation compared 
to the Control group). Ouyang et al. (2016) also reported 
that the inclusion of alfalfa flavonoids (15 mg/kg) in a diet 
intended for female broilers significantly improved BW by 
4.77%. These results are consistent with our findings.
Regarding BWG, turkeys that were given the AF-free diet 
with the addition of powdered alfalfa leaves had no statisti-
cally significant differences when compared to the Control 
group (471.56 vs 435.00 g). However, the three experimental 
groups that were fed with the diet contaminated with  AFB1 
(AF, AF+alfalfa, and AF+YCW), showed a significant reduc-
tion in BWG compared to the Control and Alfalfa groups. These 
results are similar to those reported by Samur et al. (2020), 
who showed that the supplementation of a commercial duck-
ling diet with 10% (w/w) fresh alfalfa had no effect in BWG. 
Interestingly, poults that were given the diet supplemented 
with powdered alfalfa leaves had the greatest numerical value 
in BWG compared to the Control group (but not statistically 
significant). In this context, Suwignyo et al. (2021) showed that 
3% (w/w) alfalfa supplementation in the diet increased BWG 
up to 8.05% in ducks from 1 to 35 days of age. In this research, 
the AF+alfalfa group did not present a significant difference 
in BWG compared to the Control group, and this could pos-
sibly be explained by the immuno- and hepatoprotective effect 
of the powdered alfalfa leaves. Moreover, a significant reduc-
tion in FI (g/poult) was observed in the AF and AF+YCW 
groups compared to the Control group, attaining a reduction 
of up to −16.96% and −14.49%, respectively (Table 1). These 
results agree to those reported by Rauber et al. (2007), who 
showed a −9.13% reduction in FI in turkeys fed a diet with 
200 ng  AFB1/g feed. Additionally, Singh (2019) reported a 
significant decrease (up to −34.61%) in feed consumption in 
4-week-old turkey poults, which were fed a diet with 150 μg 
 AFB1/kg feed. In this work, there was no significant difference 
in FI between the AF+alfalfa and the Control group (Table 1). 
Furthermore, it was observed that FI was significantly higher 
in the group of turkeys that were offered the  AFB1-free diet 
with the addition of the powdered alfalfa leaves (Table 1). In 
this context, Suwignyo et al. (2021) also showed that 3% (w/w) 
alfalfa supplementation in the diet increased FI and BWG of 
ducks at 35 days of age. In this study, it was observed that there 
was no statistically significant difference in the FCR between 
treatments. These results are in close agreement with Samur 
Table 1  Evaluation of body weight (BW), body weight gain (BWG), 
feed intake (FI), and feed conversion ratio (FCR) in 28-day-old tur-
key poults consuming a maize-soybean based diet contaminated with 
250  ng  AFB1/g supplemented with the powdered alfalfa leaves and 
yeast cell wall
Seven replicates per group (n = 10 poults per replicate). Means ± SE
abc Means with non-matching superscripts within columns indicates significant difference (p < 0.05)
Treatment Body weight (g) Body weight gain (g) Feed intake (g) Feed conversion ratio
Deviation from 
Control (%)
Control 491.76 ± 8.32a,b 0 435.00 ± 8.20a,b 683.68 ± 25.10a,b 1.571 ± 0.02
AF 428.14 ± 6.71c -12.94 371.76 ± 6.74c 567.76 ± 7.00c 1.533 ± 0.04
Alfalfa 528.21 ± 3.83a 7.41 471.56 ± 3.70a 726.27 ± 23.57a 1.532 ± 0.04
AF+alfalfa 470.17 ± 9.81b,c -4.40 414.61 ± 9.94b,c 607.30 ± 22.89b,c 1.474 ± 0.04
AF+YCW 441.68 ± 10.38c -10.18 385.30 ± 10.64c 584.60 ± 34.70c 1.512 ± 0.03
p-value < 0.0001 < 0.0001 < 0.001 0.16
Table 2  Relative weight of the 
liver, spleen, bursa of Fabricius 
in 28-day-old turkey poults 
consuming a maize-soybean 
based diet contaminated with 
250 ng  AFB1/g supplemented 
with powdered alfalfa leaves 
and yeast cell wall
Means ± SE
ab Means with non-matching superscripts within columns indicates significant difference (p < 0.05)
Treatment Liver Spleen Bursa of Fabricius Bursa/spleen ratio
Control 3.72 ± 0.133a 0.11 ± 0.005b 0.18 ± 0.010b 1.56 ± 0.095
AF 2.89 ± 0.127b 0.17 ± 0.009a 0.20 ± 0.010a,b 1.18 ± 0.069
Alfalfa 3.08 ± 0.121b 0.12 ± 0.005b 0.16 ± 0.007b 1.42 ± 0.081
AF+alfalfa 2.89 ± 0.134b 0.14 ± 0.009a,b 0.20 ± 0.012a,b 1.43 ± 0.115
AF+YCW 3.08 ± 0.119b 0.16 ± 0.011a 0.21 ± 0.011a 1.40 ± 0.120
p-value < 0.0001 < 0.0001 0.0042 0.10
273Mycotoxin Research (2024) 40:269–277 
et al. (2020), who reported that the consumption of a diet con-
taminated with 200 ng  AFB1/g feed does not show a significant 
difference in FCR in turkeys. Finally, the mortality observed 
during the experiment (7%) was not related to the dietary  AFB1 
level, but rather due to yolk sac infection.
Relative weight of organs
The effect of the different treatments on the relative organ’s 
weight is shown in Table 2. Compared to the Control group, the 
relative weight of the liver decreased significantly in the poults 
of the four experimental groups (AF, Alfalfa, AF+alfalfa, and 
AF+YCW). These results are consistent to those reported by 
Gómez-Espinosa et al. (2017), who observed that the relative 
weight of the liver of the poults that consumed a diet contami-
nated with  AFB1 (331 ng  AFB1/g feed) decreased significantly. 
Moreover, the group of turkeys that were offered the  AFB1-free 
diet with the addition of powdered alfalfa leaves (Alfalfa) and 
the AF+alfalfa group did not show significant differences in 
terms of the relative weight of the spleen compared to the 
Control group (Table 2). Notoriously, two of the experimental 
groups that were fed with the diet contaminated with  AFB1 (AF 
and AF+YCW), showed a significant increase on the relative 
weight of the spleen compared to the Control group (Table 2). 
Peng et al. (2015) showed that broiler chickens that were fed 
with a diet contaminated with aflatoxins (216.4 ng  AFB1/g 
feed) increased the relative weight of the spleen. Finally, the 
three groups that were fed with the diet contaminated with 
 AFB1 showed that the relative weight of the bursa of Fabricius 
increased by 11.1% (AF), 11.1% (AF+alfalfa), and 16.7% 
(AF+YCW), with respect to the Control group. Regarding the 
bursa/spleen ratio, there were no statistically significant dif-
ferences between treatments. Maguey-González et al. (2023) 
reported that the relative weight of the spleen and the bursa of 
Fabricius increased significantly in poults that were fed with a 
diet containing  AFB1 (250 ng  AFB1/g feed). These results are 
in accordance with those obtained in the present study.
Blood count cells
At the end of the trial, certain trends were observed in the blood 
cell count values; however, no significant differences were 
found in all the values analyzed such as Hct, WBCs, Ls, the 
ratio of H to Ls, Ba, Mn, and H (Table 3). These results agree 
with what was reported by Quist et al. (2000) and Oyegunwa 
et al. (2021), who studied the effect of a diet contaminated with 
200 and 150 ng  AFB1/g in turkey poults, respectively.
Selected biochemical constituents
Table  4 shows the results of some biochemical con-
stituents. In general, the plasmatic concentrations of 
Table 3  Blood count cells in 28-day-old turkey poults consuming a maize-soybean based diet contaminated with 250 ng  AFB1/g supplemented 
with powdered alfalfa leaves and yeast cell wall
Means with non-matching superscripts within columns indicates significant difference (p < 0.05). Seven replicates per group (n = 3 poults per 
replicate). Means ± SE
Hct Hematocrit, WBCs Leukocytes, Ls Lymphocytes, H/L ratio of heterophils to lymphocytes, Ba Basophils, Mn Monocytes, H heterophils
Treatment Hct WBCs H Ls H/L Ba Mn
Control 40.75 ± 3.22 15.87 ± 1.67 6421 ± 1505.83 6966 ± 492.59 0.98 ± 0.29 961 ± 298.73 1500 ± 284.19
AF 36.60 ± 3.36 20.58 ± 5.07 5169 ± 1592.10 6749 ± 1013.02 0.73 ± 0.12 650 ± 154.08 2423 ± 669.84
Alfalfa 45.66 ± 0.84 16.59 ± 1.93 5950 ± 657.23 7286 ± 890.40 0.85 ± 0.07 433 ± 203.19 2918 ± 491.72
AF+alfalfa 42.80 ± 2.54 14.26 ± 2.10 5720 ± 1018.31 5942 ± 777.85 0.94 ± 0.09 216 ± 122.07 2363 ± 792.75
AF+YCW 37.50 ± 2.20 19.90 ± 1.41 6627 ± 764.86 8795 ± 999.97 0.83 ± 0.12 784 ± 293.27 3783 ± 720.88
p-value 0.097 0.46 0.90 0.22 0.77 0.16 0.19
Table 4  Biochemical 
parameters in 28-day-old 
turkey poults consuming a 
maize-soybean based diet 
contaminated with 250 ng 
 AFB1/g supplemented with 
powdered alfalfa leaves and 
yeast cell wall
Means ± SE
TP Total protein, Glu Glucose, Ca Calcium, UA uric acid, CRE creatinine, BUN blood urea nitrogen
abc Means with non-matching superscripts within columns indicates significant difference (p < 0.05). Seven 
replicates per group (n = 3 poults per replicate)
Treatment TP Glu Ca UA CRE BUN
Control 3.82 ± 0.10a 583.00 ± 49.90a 9.04 ± 0.35a 10.80 ± 0.23 0.20 ± 0.01a 4.6 ± 0.50a,b
AF 1.68 ± 0.04b 405.00 ± 12.84b,c 5.76 ± 0.29b 9.74 ± 0.25 0.11 ± 0.01b 3.6 ± 0.24b
Alfalfa 3.88 ± 0.09a 528.20 ± 31.22a,b 8.58 ± 0.35a 10.10 ± 0.20 0.23 ± 0.01a 4.6 ± 0.24a,b
AF+alfalfa 2.00 ± 0.08b 373.00 ± 18.89c 5.74 ± 0.29b 8.50 ± 0.21 0.12 ± 0.01b 4.8 ± 0.20a
AF+YCW 1.90 ± 0.08b 382.20 ± 17.79c 4.66 ± 0.24b 9.24 ± 0.26 0.13 ± 0.01b 4.8 ± 0.20a
p-value < 0.001 0.0003 < 0.0001 0.34 < 0.0001 < 0.001
274 Mycotoxin Research (2024) 40:269–277
TP, Glu, Ca, CRE, and BUN in the poults fed with the 
 AFB1-contaminated diet significantly decreased com-
pared to the Control group. Aflatoxin consumption by 
poultry has been reported to cause a reduction in serum 
total protein levels due to impaired protein synthesis in 
the liver (Van Rensburg et al. 2006). Thus, 2.27-, 1.91-, 
and 2-fold decrease in total serum protein was found in 
the AF, AF+alfalfa, and AF+YCW groups, respectively. 
Finally, the group of poults fed the  AFB1-free diet, and 
the Alfalfa group did not present a significant difference 
in the TP concentration compared with the Control group. 
These findings are in accordance with earlier reports, 
Wan et al. (2013) reported that the amount of TP in the 
serum of ducks that consumed a diet contaminated with 
100 ng  AFB1/g feed, decreased by 1.13-fold.
In general, the groups of turkeys that were fed a 
diet contaminated with  AFB1 (AF, AF+alfalfa, and 
AF+YCW) showed a decrease in serum Glu, Ca, and CRE 
concentrations, compared to the Control group (Table 4). 
On the other hand, the group of turkeys that were offered 
the  AFB1-free diet but with the addition of the powdered 
alfalfa leaves, did not show significant differences in the 
serum concentration of Glu, Ca, and CRE. These findings 
agree with various reports with broilers (Gowda et al. 
2009; Sridhar et al. 2015; Gómez-Espinosa et al. 2017; 
Hernández-Ramírez et al. 2021). On the other hand, there 
were no significant differences in the concentration of 
UA. Sharma et al. (2019) showed that the consumption 
of a diet contaminated with 150 and 300 ng  AFB1/g feed 
in broiler chickens did not affect the UA concentration. 
Finally, the BUN value decreased only in the AF group. 
In this context, Xie et al. (2022) showed that the BUN 
value was not affected in broilers fed with two levels of 
 AFB1 (60 and 120 ng/g), at 21 and 42 days post-exposure.
Liver enzymes
Table 5 shows the results of some serum liver enzymes. 
There were no statistically significant differences between 
the five experimental groups in the serum concentrations 
of ALP, AST, CK, and GLDH. Comparable results are also 
reported by other researchers (Quist et al. 2000; Gómez-
Espinosa et al. 2017). However, the only enzyme in which a 
marked increase was observed in the AF+YCW group was 
the ALT. Lala et al. (2016) reported that ALT levels showed 
a significant increase in poultry fed with 60 ng  AFB1/g feed, 
which is consistent with our findings.
Liver histology
Table 6 and Fig. 1 show the histopathological changes in 
the liver of turkey poults. In general, no significant liver 
lesions were observed in the Control group (Fig. 1a). 
However, exposure to  AFB1 caused important changes 
in the liver of poults. For instance, a significant increase 
in the severity of liver lesions, including vacuolar 
Table 5  Liver enzyme 
concentrations in 28-day-old 
turkey poults that consumed 
a maize-soybean based diet 
contaminated with 250 ng 
 AFB1/g supplemented with 
powdered alfalfa leaves and 
yeast cell wall
Seven replicates per group (n = 3 poults per replicate). Means ± SE
ALT alanine transaminase, ALP alkaline phosphatase, AST aspartate aminotransferase, CK creatine kinase, 
GLDH glutamate dehydrogenase
ab Means with non-matching superscripts within columns indicates significant difference (p < 0.05)
Treatment ALT ALP AST CK GLDH
Control 4.80 ± 1.11b 2569.40 ± 130.07 302.60 ± 15.38 6644 ± 1567.32 11.00 ± 1.26
AF 3.00 ± 1.00b 2561.40 ± 123.31 259.00 ± 13.61 3832 ± 474.72 8.00 ± 0.44
Alfalfa 4.60 ± 1.66b 2471.40 ± 127.44 298.80 ± 25.25 5032 ± 2141.89 17.60 ± 6.09
AF+alfalfa 2.20 ± 0.20b 2159.20 ± 66.77 247.20 ± 14.01 3460 ± 738.57 7.6 ± 1.53
AF+YCW 8.80 ± 2.83a 2356.80 ± 119.92 318.80 ± 36.72 10244 ± 742.04 7.4 ± 1.12
p-value < 0.001 0.43 0.11 0.13 0.14
Table 6  Histopathological 
changes in the liver of 28-day-
old turkey poults that consumed 
a maize-soybean based diet 
contaminated with 250 ng 
 AFB1/g supplemented with 
powdered alfalfa leaves and 
yeast cell wall
Seven replicates per group (n = 3 poults per replicate). Means ± SE
ab Means with non-matching superscripts within columns indicates significant difference (p < 0.05)
Treatment Vacuolar degeneration Inflammation Bile duct hyperplasia Fibrosis
Control 1.00 ± 0.28b 0.50 ± 0.20b 0b 0b
AF 2.00 ± 0.28a 2.00 ± 0.22a 2.00 ± 0.15a 1.00 ± 0.18a
Alfalfa 1.00 ± 0.20b 0b 1.00 ± 0.11b 0b
AF+alfalfa 1.50 ± 0.27a,b 1.00 ± 0.14a,b 1.50 ± 0.09b 0b
AF+YCW 2.00 ± 0.24a 1.00 ± 0.23a,b 1.50 ± 0.18b 1.00 ± 0.11a
p-value < 0.01 < 0.0001 < 0.001 < 0.0001
275Mycotoxin Research (2024) 40:269–277 
degeneration, inflammation, bile duct hyperplasia, and 
fibrosis was observed in the AF group (Fig. 1b). Previ-
ous studies have reported histopathological alterations 
in the liver of poultry fed with  AFB1 levels ranging from 
50 to 1000 ng  AFB1/g (Giambrone et al. 1985; Sridhar 
et al. 2015; Lala et al. 2016; Hernández-Ramírez et al. 
2021; Xie et al. 2022). These alterations include vacu-
olar degeneration, fatty liver, hemorrhage, congestion, 
leukocyte infiltration, bile duct hyperplasia, hypertro-
phy, and portal fibrosis. Conversely, in the two experi-
mental groups supplemented with the powdered alfalfa 
leaves, no significant differences in the liver lesions 
were observed compared to the Control group (Table 6), 
suggesting that the lesions caused by the  AFB1 were 
significantly ameliorated when the powdered alfalfa was 
included in the  AFB1-contaminated diet. Regarding the 
Fig. 1  Comparative histological 
changes in the liver of 28-day-
old turkey poults that consumed 
a maize-soybean based diet 
contaminated with 250 ng 
 AFB1/g and supplemented with 
powdered alfalfa and yeast cell 
wall. H&E-stained tissue of: 
a Control, b AF, c Alfalfa, d 
AF+alfalfa, and e AF+YCW. 
Black circles show vacuolar 
degeneration, blue circles show 
inflammation, yellow circles 
show bile duct hyperplasia, and 
red circles show fibrosis
276 Mycotoxin Research (2024) 40:269–277
AF+YCW group, it was observed that there were no 
significant differences in inflammation and hyperplasia 
of bile ducts, compared with the Control group. How-
ever, marked vacuolar degeneration and fibrosis were 
observed (Fig. 1e). In general, the decrease in lesions in 
poults that received 0.5% (w/w) powdered alfalfa could 
be related to the effects of flavonoids, carotenoids, and 
chlorophylls contained in the leaves and mainly to its 
adsorption properties against  AFB1 (Nava-Ramírez 
et al. 2023).
To the best of our knowledge, this is the first report 
showing the effect of a low inclusion level (0.5%, w/w) 
of powdered alfalfa leaves in the diet of turkey poults con-
taminated with  AFB1 (250 ng/g) on production parameters, 
relative organ weights, total blood cell counts, biochemi-
cal and enzymatic parameters, and liver histology. In gen-
eral, the addition of the adsorbent material derived from 
powdered alfalfa leaves significantly improved the produc-
tion parameters, the relative weight of the organs, and the 
histological lesions of the liver. Moreover, the powdered 
alfalfa leaves did not cause deleterious changes in the bio-
chemical and enzymatic parameters, as well as in the total 
blood cell count. Taken together, these results allowed us 
to confirm that powdered alfalfa is an effective material 
to adsorb  AFB1 and, therefore, prevent its toxic effects in 
in vivo trials. However, it is pertinent to carry out more 
in vivo studies on the complete growing cycle of this and 
other avian species to understand the complete protective 
role of powdered alfalfa leaves and other plant-based mate-
rials with adsorptive properties against  AFB1. Research in 
this direction is in progress in our laboratories.
Acknowledgements María de Jesús Nava Ramírez acknowledges 
CONAHCYT for the scholarship CVU. 866355.
Author contribution Conceptualization: A.M.-A., C.L.-C., and A.V.-D; 
Methodology: M. d. J. N.-R., J.A.M.-G., and J.O.H.-R; Formal analysis 
and investigation: S.G.-R., and J.D.-L.; Writing—original draft prepa-
ration: M. d. J. N.-R., and A.M.-A; Writing—review and editing: X.-D., 
S.G.-R., J.A.M.-G., B.M-H., and G.T.-I; Funding acquisition: A.M.-A., 
G.T.-I., and B.M-H.; Resources: G.T.-I., and B.M-H.; Supervision: 
A.M.-A., G.T.-I., and B.M-H.
Funding This work was supported by UNAM-PAPIIT grant number 
IA101523 and by funds provided by USDA-NIFA, Sustainable Agri-
culture Systems, grant number 2019-69012-29905. Title of Project: 
Empowering U.S. Broiler Production for Transformation and Sustain-
ability USDA-NIFA.
Data availability The datasets generated during and/or analyzed dur-
ing the current study are available from the corresponding author on 
reasonable request.
Declarations 
Conflicts of interest None.
Open Access This article is licensed under a Creative Commons Attri-
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need to obtain permission directly from the copyright holder. To view a 
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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Citation: Nava-Ramírez, M.d.J.; Liu,
J.; Hernández-Ramírez, J.O.;
Hernandez-Velasco, X.; Latorre, J.D.;
Vázquez-Durán, A.; Zhang, G.;
Senas-Cuesta, R.; Gómez-Rosales, S.;
Stein, A.; et al. Exploring the Effects of
an Alfalfa Leaf-Derived Adsorbent on
Microbial Community, Ileal
Morphology, Barrier Function, and
Immunity in Turkey Poults during
Chronic Aflatoxin B1 Exposure. Int. J.
Mol. Sci. 2024, 25, 7977. https://
doi.org/10.3390/ijms25147977
Academic Editor: Hirokazu Fukui
Received: 14 May 2024
Revised: 12 July 2024
Accepted: 15 July 2024
Published: 22 July 2024
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/).
 International Journal of 
Molecular Sciences
Article
Exploring the Effects of an Alfalfa Leaf-Derived Adsorbent on
Microbial Community, Ileal Morphology, Barrier Function,
and Immunity in Turkey Poults during Chronic Aflatoxin
B1 Exposure
María de Jesús Nava-Ramírez 1 , Jing Liu 2,*, Juan Omar Hernández-Ramírez 1 , Xochitl Hernandez-Velasco 3 ,
Juan D. Latorre 4, Alma Vázquez-Durán 1, Guolong Zhang 2 , Roberto Senas-Cuesta 4 , Sergio Gómez-Rosales 5 ,
Andressa Stein 4, Billy M. Hargis 4, Guillermo Téllez-Isaías 4 , Abraham Méndez-Albores 1
and Jesús A. Maguey-González 4,*
1 Unidad de Investigación Multidisciplinaria L14 (Alimentos, Micotoxinas, y Micotoxicosis), Facultad de
Estudios Superiores (FES) Cuautitlán, UNAM, Cuautitlán Izcalli 54740, Mexico;
mari_551293@comunidad.unam.mx (M.d.J.N.-R.); mvzjohr@hotmail.com (J.O.H.-R.);
almavazquez@comunidad.unam.mx (A.V.-D.); albores@unam.mx (A.M.-A.)
2 Department of Animal and Food Sciences, Oklahoma State University, Stillwater, OK 74078, USA;
glenn.zhang@okstate.edu
3 Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia, UNAM,
Ciudad de México 04510, Mexico; xochitlh@fmvz.unam.mx
4 Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; jl11@uark.edu (J.D.L.);
rsenascu@uark.edu (R.S.-C.); andressastein.s@gmail.com (A.S.); bhargis@uark.edu (B.M.H.);
gtellez@uark.edu (G.T.-I.)
5 Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal (CENID-INIFAP), Km 1
Carretera a Colon, Ajuchitlán, Querétaro 76280, Mexico; gomez.sergio@inifap.gob.mx
* Correspondence: jing.liu12@okstate.edu (J.L.); jm201@uark.edu (J.A.M.-G.)
Abstract: This article follows-up on our recently published work, which evaluated the impact of the
addition of an alfalfa leaf-derived adsorbent in the aflatoxin B1 (AFB1)-contaminated diet in regard to
the production parameters, blood cell count, serum biochemistry, liver enzymes, and liver histology of
turkey poults. This paper presents complementary results on microbial community, ileal morphology,
barrier function, and immunity. For this purpose, 350 1-day-old female turkey poults were randomly
distributed into five groups: (1) Control, AFB1-free diet; (2) AF, AFB1-contaminated diet at 250 ng/g;
(3) alfalfa, AFB1-free diet + 0.5% (w/w) adsorbent; (4) alfalfa + AF, AFB1-contaminated diet at
250 ng/g + 0.5% (w/w) adsorbent; and (5) YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5%
(w/w) commercial yeast cell wall-based adsorbent (reference group). In general, in the AF group, the
growth of opportunistic pathogens was promoted, which lead to gut dysbacteriosis, mainly influenced
by Streptococcus lutetiensis. Conversely, a significant increase in beneficial bacteria (Faecalibacterium
and Coprococcus catus) was promoted by the addition of the plant-based adsorbent. Moreover, the AF
group had the lowest villus height and a compromised barrier function, as evidenced by a significant
(p < 0.05) increase in fluorescein isothiocyanate dextran (FITC-d), but these negative effects were
almost reversed by the addition of the alfalfa adsorbent. Furthermore, the AF + YCW and alfalfa + AF
groups exhibited a significant increase in the cutaneous basophil hypersensitivity response compared
to the rest of the experimental groups. Taken together, these results pointed out that the alfalfa
counteracts the adverse effects of AFB1 in poults, facilitating the colonization of beneficial bacteria
and improving the barrier function of the turkey poults.
Keywords: aflatoxin B1; alfalfa; cellular immunity; gut integrity; microbiota; turkey poults
Int. J. Mol. Sci. 2024, 25, 7977. https://doi.org/10.3390/ijms25147977 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2024, 25, 7977 2 of 13
1. Introduction
Aflatoxin B1 (AFB1) is a potent secondary metabolite mainly synthesised by Aspergillus
spp. AFB1 contamination in poultry feed negatively influences gut health, immunity, and
performance. As a result, AFB1 contamination has become a major preoccupation in the
world of poultry producers and researchers [1]. Consequently, to avoid these adverse
effects, multiple strategies have been developed to control AFB1 in feed and to improve the
health of broiler birds in the poultry industry. Recently, functional natural feed additives
have been considered as promising substitutes for antibiotic growth promoters due to their
antioxidant, anti-inflammatory, and immune-modulation properties [2].
While maize and soy remain the foundation of many (if not most) broiler diets, sup-
plementation with nutrient-dense forages such as alfalfa could offer several advantages.
Packed with vitamins, minerals, and bioactive compounds, alfalfa stands to enhance the
environmental profile of broiler production while promoting the health and welfare of
the chicken itself. First, alfalfa is nutritious, its protein content is high, and, as such, it is
an excellent building block for muscle. Moreover, vitamins A, E, and K, along with its
calcium, magnesium, and iron content, provide health support and promote strong and
healthy bones [3]. Alfalfa’s carotene content also contributes to the yellow colouration of
the chicken skin and yolk, something that is linked to important economic preference in
certain consumer markets [4]. Concerning gut health, alfalfa’s high fibre content stimulates
the production of beneficial gut bacteria, helping to keep the chicken healthy and enhance
their efficiency [5]. In this context, some researchers have also suggested the possibility of
having higher meat quality when alfalfa is included in broiler diets [6]. Moreover, the high
level of natural antioxidants contributes to increasing the shelf life of the meat as well as
reducing lipid oxidation [7].
Feeding alfalfa also brings about environmental benefits. The nitrogen-fixing ability
of alfalfa reduces fertiliser application in a pasture, while its deep roots improve soil health
and the retention of water [8]. These benefits can be translated to lower environmental
impact and higher sustainability during broiler production.
The outcomes from the recent in vitro [9] and in vivo [10] studies from our labora-
tories confirmed the efficacy of alfalfa in ameliorating AFB1 toxicity and enhancing the
performance in turkey poults. The present study continues the same experimental design
to determine the effects of alfalfa on the composition and diversity of the gut microbial
community, intestinal permeability, the morphometric analysis of ileum, and the cellular
immunity. This research complements and indicates crucial roles of alfalfa leaves in the
protection against aflatoxin B1-induced toxicity, suggesting that this intervention can be
used as a novel approach to improve intestinal health and enhance resilience in turkey
poults during aflatoxicosis.
2. Results
There were no obvious distinctions among the experimental groups in terms of α-
diversity (Figure 1). Nevertheless, the AF group exhibited a lower diversity (number of
observed ASVs, Evenness, and Shannon indexes) than the Control group, indicating that the
challenge to AFB1 could potentially result in the proliferation of specific pathogenic bacteria,
which could contribute to a change in the bacterial community. However, supplementation
of alfalfa could almost restore this decreased trend of bacteria to healthy levels (Figure 1).
Similarly, there was no significant separation among groups based on the weighted
UniFrac distance (Figure 1). However, an unweighted UniFrac distance showed a significant
difference among the Control and AF groups, indicating the influence of AFB1 on the
microbial community composition (p < 0.05). In addition, a clear separation between the
AF group and the AF + YCW group was also observed (p = 0.017, R2 = 0.168), suggesting
that YCW supplementation during the AFB1 challenge could also change the bacterial
community structure (β-diversity).
Int. J. Mol. Sci. 2024, 25, 7977 3 of 13
Figure 1. Alpha and beta diversities of the cecal microbiota among different experimental groups. 
The cecal contents (n = 7/treatment) were subjected to 16S rRNA gene sequencing. Observed ASV, 
Pielou’s Evenness, and Shannon Index were calculated to measure the α-diversity of the cecal mi-
crobiota. Kruskal–Wallis test was used for statistical significance determination. The β-diversity-
weighted UniFrac and unweighted UniFrac distances were used to generate the principal coordi-
nates analysis (PCoA) plots. Permutational multivariate analysis of variance (PERMANOVA) was 
used for statistical significance determination. Control, AFB1-free diet; AF, AFB1-contaminated diet 
at 250 ng/g; alfalfa, AFB1-free diet + 0.5% (w/w) adsorbent; alfalfa + AF, AFB1-contaminated diet at 
250 ng/g + 0.5% (w/w) adsorbent; and YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) 
commercial yeast cell wall-based adsorbent (reference group).
Table 1 summarises the findings of the relative abundances (%) of the cecal bacterial 
phyla and their families between the experimental groups. The contamination with 250 
ng AFB1/g did not affect the phyla relative abundances of the microbial community in the 
caecum of any of the experimental groups. However, at the family level, a significant de-
crease in Streptococcaceae populations was observed in the Control group compared to the 
rest of the experimental groups (p < 0.04). In addition, the relative genera abundance of 
Coprobacillaceae was reduced in the AF and alfalfa + AF groups, and the same pattern was 
observed in the ASV levels (p < 0.02). On the other hand, the alfalfa group exhibited a 
substantial increase in Faecalibacterium levels in contrast to the other experimental groups 
(Table 2). At the ASV level, bacterial biomarkers for each group were identified using lin-
ear discriminant analysis effect size (LEfSe), employing an all-against-all multiclass anal-
ysis approach (Figure 2). Several bacterial species were found to be enriched in the LEfSe 
analysis using the data from all experimental groups. For instance, the signature ASVs for 
the Control group were Mediterraneibaer (F91) and Lachnospiracaeae unidentified (F25), 
while AF group had greater abundances of Streptococcus lutetiensis (F6). In addition, the 
alfalfa group were Faecalibacterium (F7 and F53) and Coprococcus catus (F85), while the al-
falfa + AF group had greater abundances of Bacillus (F8) and Anaerotignum (F99).
Figure 1. Alpha and beta diversities of the cecal microbiota among different experimental groups. The
cecal contents (n = 7/treatment) were subjected to 16S rRNA gene sequencing. Observed ASV, Pielou’s
Evenness, and Shannon Index were calculated to measure the α-diversity of the cecal microbiota.
Kruskal–Wallis test was used for statistical significance determination. The β-diversity-weighted
UniFrac and unweighted UniFrac distances were used to generate the principal coordinates analysis
(PCoA) plots. Permutational multivariate analysis of variance (PERMANOVA) was used for statistical
significance determination. Control, AFB1-free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa,
AFB1-free diet + 0.5% (w/w) adsorbent; alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5%
(w/w) adsorbent; and YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial
yeast cell wall-based adsorbent (reference group).
Table 1 summarises the findings of the relative abundances (%) of the cecal bacterial
phyla and their fa ilies between the experimental groups. The contamination with 250 ng
AFB1/g did not affect the phyla relative abundances of the microbial community in the
caecum of any of the experimental groups. However, at the family level, a significant
decrease in Streptococcaceae populations was observed in the Control group compared to
the rest of the experimental groups (p < 0.04). In addition, the relative genera abundance
of Coprobacillaceae was reduced in the AF and alfalfa + AF groups, and the same pattern
was observed in the ASV levels (p < 0.02). On the other hand, the alfalfa group exhibited a
substant al increase in Faecalibacterium levels in contrast to the other experimental grou s
(Table 2). At the ASV level, bacterial biomarkers for each group were identified using linear
discriminant analysis effect size (LEfSe), employing an all-against-all multiclass analysis
approach (Figure 2). Several bacterial species were found to be enriched in the LEfSe
analysis using the data from all experimental groups. For instance, the signature ASVs for
the Control group were Mediterraneibaer (F91) and Lachnospiracaeae unidentified (F25), while
AF group had greater abundances of Streptococcus lutetiensis (F6). In addition, the alfalfa
group were Faecalibacterium (F7 nd F53) and Coprococc s catus (F85), while the alfalfa + AF
group had greater abundances of Bacillus (F8) and Anaerotignum (F99).
Int. J. Mol. Sci. 2024, 25, 7977 4 of 13
Table 1. Relative abundances (%) of the cecal bacterial phyla and families among the experimental
groups.
Taxon Control AF Alfalfa Alfalfa + AF AF + YCW SEM p-Value
Phyla
Firmicutes 79.42 74.04 81.02 82.45 72.78 1.92 0.13
Proteobacteria 14.10 20.76 16.52 12.93 20.82 1.64 0.17
Tenericutes 3.85 2.84 1.11 2.94 4.57 0.58 0.45
Cyanobacteria 0.78 1.45 0.08 0.26 0.02 0.26 0.62
Actinobacteria 0.45 0.08 0.08 0.42 0.15 0.08 0.39
Families
Oscillospiraceae 32.72 32.53 40.05 39.90 30.04 2.06 0.06
Lachnospiraceae 32.28 25.69 28.66 28.64 28.21 1.05 0.15
Enterobacteriaceae 14.10 20.75 16.52 12.89 20.82 1.64 0.17
Clostridiales_unidentified 4.85 5.97 4.11 3.29 4.05 0.45 0.24
Erysipelotrichaceae 3.81 1.70 4.74 2.54 4.04 0.54 0.09
Bacillaceae 1.55 1.47 0.52 4.20 2.12 0.61 0.06
Mollicutes_unidentified 3.04 2.56 0.81 2.19 4.01 0.52 0.30
Lactobacillaceae 1.67 1.84 0.36 1.51 1.74 0.27 0.10
Christensenellaceae 0.76 0.81 0.80 0.93 1.23 0.08 0.68
Streptococcaceae 0.09 b 0.97 a 1.09 a 0.71 a 0.45 a 0.18 0.04
Vampirovibrio_unidentified 0.78 1.45 0.08 0.26 0.02 0.26 0.62
Enterococcaceae 0.67 0.14 0.10 0.20 0.23 0.10 0.18
Clostridia_unidentified 0.14 0.85 0.03 0 0 0.16 0.58
Peptostreptococcaceae 0.41 0.29 0.09 0.13 0.09 0.06 0.12
Clostridiaceae 1 0.25 0.29 0.19 0.12 0.19 0.02 0.69
The mean relative abundances (%) of top five phyla and top 15 families of cecal microbiota of different groups
are shown means (n = 7/treatment). a,b indicates significant differences between the treatments within the rows
(p < 0.05). Statistical as significance was determined using the Kruskal–Wallis test, followed by the pairwise
Wilcoxon rank sum test. Control, AFB1-free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa, AFB1-free
diet + 0.5% (w/w) adsorbent; alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; and
YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based adsorbent
(reference group).
Table 2. Relative abundances (%) of the cecal bacterial genera and ASVs among the experimental
groups.
Taxon Control AF Alfalfa Alfalfa + AF AF + YCW SEM p-Value
Genera
Escherichia/Shigella 13.16 20.71 16.37 12.76 20.67 1.73 0.17
Oscillospiraceae_unidentified 10.21 11.14 10.97 14.11 11.05 0.67 0.68
Mediterraneibacter 10.54 7.50 8.76 9.49 8.96 0.49 0.62
Lachnospiraceae_unidentified 6.27 6.07 6.29 7.30 6.83 0.22 0.69
Subdoligranulum 8.70 4.93 7.29 6.65 3.03 0.98 0.59
Pseudoflavonifractor 5.04 5.91 4.36 7.05 5.76 0.45 0.32
Enterocloster 4.64 3.54 5.24 5.05 3.99 0.32 0.48
Clostridiales_unidentified 4.85 5.97 4.11 3.29 4.05 0.45 0.24
Blautia 4.24 2.67 2.36 1.56 2.37 0.44 0.21
Bacillus 1.55 1.47 0.52 4.20 2.12 0.61 0.06
Faecalibacterium 0.18 0.26 7.70 0.37 0.66 1.46 0.07
Coprobacillaceae_unidentified 1.72 ab 0.44 b 3.06 a 0.69 b 2.24 a 0.48 0.02
Mollicutes_unidentified 3.04 2.56 0.81 2.19 4.01 0.52 0.30
Anaerostipes 2.51 0.75 1.35 1.43 1.33 0.28 0.11
Eisenbergiella 1.50 1.55 1.23 0.78 1.44 0.14 0.46
Int. J. Mol. Sci. 2024, 25, 7977 5 of 13
Table 2. Cont.
Taxon Control AF Alfalfa Alfalfa + AF AF + YCW SEM p-Value
ASVs
Escherichia/Shigella_F1 12.8 20.24 15.86 12.54 20.23 1.69 0.14
Subdoligranulum_variabile_F4 5.11 1.88 3.80 3.36 1.93 0.60 0.81
Mediterraneibacter_F2 2.65 2.02 1.37 2.54 3.79 0.39 0.25
Mediterraneibacter_F3 2.14 3.31 3.41 1.02 2.03 0.44 0.35
Enterocloster_F5 2.65 1.25 2.56 2.08 2.06 0.24 0.44
Bacillus_F8 1.55 1.47 0.52 4.20 2.12 0.61 0.06
Mollicutes_unidentified_F9 2.77 1.19 0.64 0.80 3.30 0.54 0.50
Blautia_obeum_F18 2.93 1.67 1.34 1.07 1.31 0.33 0.82
Coprobacillaceae_unidentified_F13 1.72 ab 0.44 b 3.06 a 0.69 b 2.24 a 0.48 0.02
Enterocloster_F11 1.24 1.36 1.51 1.68 1.04 0.10 0.79
Pseudoflavonifractor_capillosus_F16 0.93 1.22 1.03 1.30 2.43 0.27 0.45
Pseudoflavonifractor_F14 1.10 1.57 1.17 1.48 1.29 0.08 0.57
Oscillospiraceae_unidentified_F17 0.80 0.92 1.06 1.39 1.96 0.20 0.98
Oscillospiraceae_unidentified_F23 2.08 0.33 0.78 1.09 1.73 0.31 0.30
Pseudoflavonifractor_F20 1.35 1.14 0.55 2.06 0.44 0.29 0.21
Butyricicoccus_pullicaecorum_F24 1.29 0.13 1.72 1.98 0.47 0.35 0.68
Anaerostipes_butyraticus_F19 1.77 0.56 1.20 0.71 1.10 0.21 0.18
Faecalibacterium_F7 0.13 b 0.24 b 4.60 a 0.34 b 0.36 b 0.86 0.02
Pseudoflavonifractor_F21 0.82 1.07 0.73 1.25 1.23 0.10 0.60
Oscillibacter_F15 0.92 1.25 0.58 1.30 0.91 0.13 0.73
The mean relative abundances (%) of top 15 genera, and top 20 ASVs of cecal microbiota of different groups are
shown as means (n = 7/treatment). a,b indicates significant differences between the treatments within the rows
(p < 0.05). Statistical significance was determined using the Kruskal–Wallis test, followed by the pairwise Wilcoxon
rank sum test. Control, AFB1-free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa, AFB1-free diet + 0.5%
(w/w) adsorbent; alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; and YCW + AF,
AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based adsorbent (reference group).
Figure 2. Differential enrichment of bacterial ASVs between different experimental groups (n = 
7/treatment) was determined using linear discriminant analysis (LDA) effect size (LEfSe), with the 
all-against-all multiclass analysis, p < 0.05, and a logarithmic LDA threshold of 3.0. Control, AFB1-
free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa, AFB1-free diet + 0.5% (w/w) adsorbent; 
alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; and YCW + AF, AFB1-
contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based adsorbent (reference 
group).
Figure 3. Relative abundances of differentially enriched bacterial ASVs (n = 7/treatment). Signifi-
cance was calculated using Kruskal–Wallis test. a,b indicates significant differences between treat-
ments (p < 0.05). Control, AFB1-free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa, AFB1-free 
diet + 0.5% (w/w) adsorbent; alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; 
and YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based 
adsorbent (reference group).
Figure 2. Differential enrichment of bacterial ASVs between different experimental groups
(n = 7/treatment) was determined using linear discriminant analysis (LDA) effect size (LEfSe), with
the all-against-all multiclass analysis, p < 0.05, and a logarithmic LDA threshold of 3.0. Control,
AFB1-free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa, AFB1-free diet + 0.5% (w/w) ad-
sorbent; alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; and YCW + AF,
AFB1-contam nated diet at 25 ng/g + 0.5% (w/w) commercial yeast cell wall- ased adsorb nt
(reference group).
In addition, Figure 3 shows the relative abundances of differentially enriched bacterial
ASVs. Alfalfa administration led to a substantial increase in Faecalibacterium and C. catus
(p < 0.05). Moreover, the populations of Bacillus and Anaerotignum, as well as microorgan-
isms related to beneficial bacteria populations and producers of short-chain fatty acids
(SCFAs), exhibited a substantial increase in poults that were fed with alfalfa and challenged
Int. J. Mol. Sci. 2024, 25, 7977 6 of 13
with AFB1 (p < 0.05). On the other hand, the results of the morphometric analysis, serum
levels of FITC-d, and cutaneous basophil hypersensitivity response (CBH) are summarised
in Table 3. In general, the AF group had the lowest villus height (p < 0.0001) and the total
villus cross-sectional area (p < 0.0001). Meanwhile, the Control (780.0 µm), AF + YCW
(700.0 µm), alfalfa (693.3 µm), and alfalfa + AF (611.9 µm) groups showed a significant
increase in villus height, followed by the AF (350.6 µm) group. Similarly, a significant
increase in the total villus cross-sectional area was observed in the Control (103.2 µm2),
AF + YCW (74.1 µm2), alfalfa (71.7 µm2), and alfalfa + AF (63.6 µm2) groups, followed by
the AF (44.7 µm2) group (Figure 4). Furthermore, the AF group had the greatest serum
FITC-d concentration (p < 0.007) in comparison to the other experimental groups. Moreover,
poults of the AF + YCW (0.96 mm) and alfalfa + AF (0.86 mm) groups showed a significant
increase in CBH response (p < 0.0001) in comparison to the rest of the experimental groups.
Figure 2. Differential enrichment of bacterial SVs between different experimental groups (n = 
7/treatment) was determined using linear discriminant analysis (LDA) effect size (LEfSe), with the 
all-against-all multiclass analysis, p < 0.05, and a logarithmic LDA threshold of 3.0. Control, AFB1-
free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa, AFB1-free diet + 0.5% (w/w) adsorbent; 
alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; and YCW + AF, AFB1-
contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based adsorbent (reference 
group).
Figure 3. Relative abundances of differentially enriched bacterial ASVs (n = 7/treatment). Signifi-
cance was calculated using Kruskal–Wallis test. a,b indicates significant differences between treat-
ments (p < 0.05). Control, AFB1-free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa, AFB1-free 
diet + 0.5% (w/w) adsorbent; alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; 
and YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based 
adsorbent (reference group).
Figure 3. Relative abundances of differentially enriched bacterial ASVs (n = 7/treatment). Significance
was calculated using Kruskal–Wallis test. a,b indicates significa t differenc s between tr atments
(p < 0.05). Control, AFB1-free diet; AF, B1-contaminated diet a 250 ng/g; alfalfa, AFB1-fr
i t . ( w) adsorbent; alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) ad-
sorbent; and YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell
wall-based adsorbent (reference group).
Table 3. Effect of powdered alfalfa leaves on ileum morphometric analysis §, serum levels of
FITC-d ¥, and cutaneous basophil hypersensitivity response (CBH) in turkey poults consuming a
maize–soybean-meal-based diet contaminated with 250 ng AFB1/g for 28 days.
Control AF Alfalfa Alfalfa + AF AF + YCW SEM * p-Value
Villus height (µm) 780.0 c 350.6 a 693.3 bc 611.9 b 700.0 bc 271.8 <0.0001
Villus width (µm) 118.9 a 146.3 b 108.8 a 112.7 a 116.9 a 36.9 0.02
Total area (µm2) 103.2 c 44.7 a 71.7 b 63.6 b 74.1 b 33.3 <0.001
FITC-d (ng/mL) 263.3 b 858.2 a 214.8 b 213.5 b 230.7 b 654.7 0.007
CBH (mm) 0.37 b 0.50 b 0.53 b 0.86 a 0.96 a 0.23 <0.0001
a,b,c Means with non-matching superscripts within rows indicates significant difference at p < 0.05. § Sixty-three
measurements were taken per variable (In each treatment). ¥ Seven replicates/group (n = 1 poults per replicate).
* Standard error of the mean. Control, AFB1-free diet; AF, AFB1-contaminated diet at 250 ng/g; alfalfa, AFB1-
free diet + 0.5% (w/w) adsorbent; alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent;
and YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based adsorbent
(reference group).
Int. J. Mol. Sci. 2024, 25, 7977 7 of 13
Table 3. Effect of powdered alfalfa leaves on ileum morphometric analysis §, serum levels of FITC-
d ¥, and cutaneous basophil hypersensitivity response (CBH) in turkey poults consuming a maize–
soybean-meal-based diet contaminated with 250 ng AFB1/g for 28 days.
Villus height (μm)
(μm)
Total area (μm
a,b,c Means with non-matching superscripts within rows indicates significant difference at p < 0.05. § 
Sixty-three measurements were taken per variable (In each treatment). ¥ Seven replicates/group (n 
= 1 poults per replicate). * Standard error of the mean. Control, AFB1-free diet; AF, AFB1-contami-
nated diet at 250 ng/g; alfalfa, AFB1-free diet + 0.5% (w/w) adsorbent; alfalfa + AF, AFB1-contami-
nated diet at 250 ng/g + 0.5% (w/w) adsorbent; and YCW + AF, AFB1-contaminated diet at 250 ng/g 
+ 0.5% (w/w) commercial yeast cell wall-based adsorbent (reference group).
Figure 4. Morphometric analysis of the ileum in turkey poults fed a diet containing AFB1 and ad-
sorbent materials. Histological images were taken using a 4× objective on H&E-stained tissue sec-
tions. (A) Control, AFB1-free diet; (B) AF, AFB1-contaminated diet at 250 ng/g; (C) alfalfa, AFB1-free 
diet + 0.5% (w/w) adsorbent; (D) alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) ad-
sorbent; and (E) YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell 
wall-based adsorbent (reference group). L = Large (μm); W = Width (μm); A = Area (μm2).
3. Discussion
In this work, the inclusion of powdered alfalfa leaves into the AFB1-contaminated 
feed confirmed the adsorbent properties of this material previously described in in vitro 
[9] and in vivo [10] assays. AFB1-contaminated feed may lower the growth rate and feed 
efficiency of poultry. However, due to the high protein content and balanced mineral com-
position, alfalfa may partly alleviate these adverse effects by supporting optimal growth 
for turkey poults. Thus, the adsorbent properties of alfalfa against AFB1 explain the reason 
for having improved growth of poults challenged with AFB1 [10]. Furthermore, alfalfa 
Figure 4. Morphometric analysis of the ileum in turkey poults fed a diet containing AFB1 and
adsorbent materials. Histological images were taken using a 4× objective on H&E-stained tissue
sections. (A) Control, AFB1-free diet; (B) AF, AFB1-contaminated diet at 250 ng/g; (C) alfalfa, AFB1-
free diet + 0.5% (w/w) adsorbent; (D) alfalfa + , AFB1-contaminated diet at 250 ng/g + 0.5% (w/w)
adsorbent; and (E) YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast
cell wall-based adsorbent (reference group). L = Large (µm); = Width (µm); A = Area (µm2).
3. iscussion
In this work, the inclusion of powdered alfalfa leaves into the AFB1-contaminated feed
confirmed the adsorbent properties of this material previously described in in vitro [9] and
in vivo [10] assays. AFB1-contaminated feed may lower the growth rate and feed efficiency
of poultry. However, due to the high protein content and balanced mineral composition,
alfalfa may partly alleviate these adverse effects by supporting optimal growth for turkey
poults. Thus, the adsorbent properties of alfalfa against AFB1 explain the reason for having
improved growth of poults challenged with AFB1 [10]. Furthermore, alfalfa provides
vitamins, minerals, fibre, and proteins that enhance chickens’ diets and favour wellness and
overall performance. Notably, the natural antioxidants present in alfalfa, such as vitamins
A, C, and E, help to prevent the oxidative stress induced by AFB1 [11]. Indeed, AFB1 could
generate a series of reactive oxygen species, and antioxidants in alfalfa might increase the
ability of broilers to counteract oxidative damage. In addition, alfalfa contains a diverse
array of immune-boosting compounds [12]. By boosting the immune system, alfalfa helps
broiler chickens manage the immunosuppressive effects of AFB1 [13]. It is well known
that AFB1 primarily attacks the liver when administered to poultry [14], causing increased
liver damage while lowering the antioxidant status in the liver tissue and plasma. Alfalfa’s
potential detoxification properties and nutrient profile may prove useful in maintaining
liver function and preventing the damage of the liver from toxins such as AFB1 [15].
In the present study, the LEfSE analysis likely identified S. lutetiensis as the most
abundant bacterial species in the AFB1-treated group. This finding suggests that the
presence of AFB1 improves the proliferation of S. lutetiensis in the gut of turkey poults.
The toxic effects of AFB1 can affect both cell-mediated and humoral immunity in poultry,
thus facilitating the entry and invasion of bacterial pathogens [16]. Hence, the mycotoxin-
induced immunosuppression promotes the colonization of opportunistic pathogens like S.
Int. J. Mol. Sci. 2024, 25, 7977 8 of 13
lutetiensis in the gut of turkey poults [17,18]. In addition, AFB1 has been reported to cause
dysbacteriosis [19] and impair the nutrient absorption capacity, weaken their immune
system, and generate a more suitable environment for S. lutetiensis growth [20].
On the other hand, the LEfSE analysis also showed an enhanced abundance of Faecal-
ibacterium within the alfalfa-fed group compared with the positive control group (poults
that received the AFB1), possibly due to the intricate interactions between alfalfa’s prop-
erties and AFB1’s toxicity. Alfalfa has plenty of bioactive compounds, such as saponins
and phenolic acids, which have the potential to bind AFB1 within the gut, thus reducing
its absorption and minimising its toxicity [21]. This finding suggests that Faecalibacterium,
rather than being inhibited by the AFB1 challenge, has flourished within a friendlier gut
environment. Furthermore, alfalfa is replete with dietary fibres, particularly oligosaccha-
rides and fructooligosaccharides (FOS), acting as prebiotics—substances that stimulates the
growth and activity of a specific, beneficial gut bacteria—such as Faecalibacterium [22]. FOS
have also been reported to exhibit antimicrobial activity by prohibiting pathogenic microbes
from adhering to the intestinal epithelium. Furthermore, alfalfa demonstrates antioxidant
potency, most notably by carotenoids and phenolic acids [23]. These antioxidants battle
the oxidative stress that stems from AFB1 and, consequently, promote friendlier gut envi-
ronments for Faecalibacterium growth [21]. Interestingly, a recent study has demonstrated a
natural resilience of Faecalibacterium towards AFB1 [24] while also documenting the pro-
duction of anti-inflammatory metabolites, especially butyrate, which can help ameliorate
the gut inflammation linked to AFB1 [25]. The combination of Faecalibacterium prolifer-
ation with those anti-inflammatory metabolites may in turn facilitate the proliferation
of other health-promoting microbes and limiting pathogenic microbes by reducing their
growth and their ability to colonise the gastrointestinal tract [26]. Taken together, the
competitive microbial advantage, in combination with the changes to the gut environment
through alfalfa supplementation, could add to the explanation for the Faecalibacterium’s
increased abundance.
Another notation worthy of mentioning by the LEfSE analysis is the increased relative
abundance of C. catus in turkey poults that received alfalfa and AFB1. C. catus belongs to
the class Clostridia, order Eubacteriales. Recently, investigators have noticed favourable
correlations between C. catus and health [27]. Consequently, it is plausible to speculate that
the increased abundance in C. catus in the turkey poults that received the diet contaminated
with AFB1 might be attributed to the capability of the bacterium to resist and metabolise
AFB1 [28]. This in turn will be a selective advantage for it over other bacteria, which
succumb to the toxic effects of AFB1 [29]. Interestingly, exposure of prokaryotes and
eukaryotes to AFB1 has been documented as a cause of stress [30]. C. catus has been shown
to exhibit enhanced stress tolerance in comparison with other gut microbiota [31], which
might afford it superior survival in the gut stressed by AFB1 and ultimately increase its
relative abundance.
The notable reduction in the serum concentration of FITC-d in turkey poults fed with
alfalfa and AFB1 compared with the positive control group may be influenced by several
factors. For instance, AFB1 can induce intestinal inflammation and increase intestinal
permeability [32]. On the other hand, alfalfa is rich in bioactive compounds such as tannins
and phenolic acids, which are well known for their binding properties against AFB1 in
the digestive tract, particularly in the intestine, reducing its absorption [33]. Consequen-
tially, this should result in a lower serum concentration of FITC-d. Moreover, alfalfa is
rich in antioxidants, mainly expressed by the presence of chlorophyll, carotenoids, and
vitamin E ([34]. These bioactive compounds have the capability of scavenging free radicals
generated during the AFB1 metabolism and, ultimately, reducing the degree of oxidative
damage, which could be responsible for the translocation of FITC-d, by affecting intestinal
epithelial cells [34]. Overall, a better intestinal barrier will contribute to a better intestinal
integrity. Furthermore, alfalfa can also improve the performance of the gastrointestinal
tract by augmenting populations of beneficial bacteria and suppressing those considered
harmful [6]. The shift from ‘harmful’ to ‘beneficial’ gut microbiota will be essential in
Int. J. Mol. Sci. 2024, 25, 7977 9 of 13
increasing the efficiency of bacteria responsible for AFB1 degradation, as well as for com-
peting with the mycotoxin for binding sites in the intestine, all of which will ultimately
decrease its bioavailability. Alternatively, the immunomodulatory action of alfalfa [35] and
its anti-inflammatory properties [36] could contribute to the epithelial tissue repair in the
intestinal epithelium and, therefore, reduce the FITC-d leakage.
In addition, the increase in ileum villus height in the AFB1-treated turkey poults with
the addition of alfalfa as compared with the Control birds is quite interesting, but the
exact mechanism is complex. AFB1 is a very potent hepatotoxin; however, exposure to this
mycotoxin can also impair functions of the intestine, which can result in damage to the
lining of villus where the enterocytes are situated. This damage can result in a reduction
in the capacity of the body to absorb nutrients and can also have an impact on the overall
health of the gut. Despite the damage to the enterocytes lining the villus, the body might
try to compensate for this damage by increasing the height of the villus in order to maintain
a greater surface area for absorption. This effect could possibly be viewed as a mechanism
to help the body adapt over the short-term exposure. Moreover, fibres and other bioactive
compounds from alfalfa could be prebiotic to beneficial gut bacteria, thus promoting their
growth and maintaining the health of the gut and the absorption of nutrients [37]. For
instance, increasing beneficial bacteria and their metabolites could stimulate villus growth
and improve intestinal function. The antioxidants present in alfalfa might also reduce
oxidative stress because of AFB1, which, in turn, could be beneficial in regard to healthier
villus [23].
4. Materials and Methods
4.1. Animal Source, Diets, and Experimental Design
Three hundred and fifty 1-day-old female Nicholas-700 turkey poults (Aviagen Inc.,
Huntsville, AR, USA) were raised in pens for 28 d. Yeast cell wall (YCW) was used as a
reference material because it is a commercial mycotoxin binder. Poults were collectively
weighted (10 birds/pen) with 7 repetitions per treatment and randomly allocated to one
of the five experimental groups: (1) Control, AFB1-free diet; (2) AF (aflatoxin), AFB1-
contaminated diet at 250 ng/g; (3) alfalfa (alfalfa adsorbent), AFB1-free diet + 0.5% (w/w)
adsorbent; (4) alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent;
and (5) YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast
cell wall-based adsorbent. The percentage of adsorbents added to the diets is equivalent to
5 g of adsorbent/kg of feed consumed. Details of the experimental diet contaminated with
AFB1 and the adsorbent material are fully described in our previous study [9]. Water and
feed were offered ad libitum. After 28 d, from each replication, three poults (n = 21) were
selected to evaluate gut integrity and cellular immunity. One poult from each replication
(n = 7) was also selected to assess the morphometry of the ileum and ceca content for
the microbiota analysis. All animal handling procedures complied with the Institutional
Animal Care and Use Committee (IACUC) at the University of Arkansas, Fayetteville
(protocol No. 22020).
4.2. Microbial Community
Following euthanasia, one cecum was removed, and the contents were manually
squeezed into a sterile tube featuring a RNA/DNA shield (Zymo Research, Irvine, CA,
USA). Samples were stores at −20 ◦C until DNA extraction was performed. A Quick-DNA
Fecal/Soil Mi-crobe Miniprep Kit (Zymo Research, Irvine, CA, USA) was used to extract
microbial DNA from the ceca contents following the manufacturer’s instructions. With
the NanoDrop ND-1000 (Wilmington, DE, USA), the DNA concentration and quality were
assessed. The bacterial 16S rRNA gene’s V3–V4 region was amplified using primers 341F
(CCTAYGGGRBGCASCAG) and 806R (GGACTAC-NNGGGTATCTAAT). A NEBNext®
UltraTM Library Prep Kit (New England Biolabs, Ipswich, MA, USA) was used to produce
a library, which was then sequenced using PE250 on an Illumina HiSeq platform.
Int. J. Mol. Sci. 2024, 25, 7977 10 of 13
4.3. Bioinformatics and Statistical Analysis
The QIIME 2 pipeline (v. 2023.07) was employed to analyse raw DNA sequencing reads.
In summary, the cut–adapt plugin was used to eliminate primer and adapter sequences
from every read. After that, low-quality reads were eliminated using the quality filter
and paired-end reads were combined using VSEARCH join pairs. Sequences were then
trimmed to 403 nucleotides and denoised by Deblur [38]. The resultant amplicon sequence
variants (ASVs) were then classified using a Bayesian classifier and the Ribosomal Database
Project (RDP) 16S rRNA training set (v. 18) for the bacterial taxonomy. For categorization, a
bootstrap confidence of 80% was applied. ASVs with a classification of <80% were assigned
the name of the last confidently assigned level followed by “unclassified”. The analysis
excluded samples with ASVs found in less than 5% of the samples. The EzBioCloud 16S
database (v.2023.08.23. https://www.ezbiocloud.net/identify Accessed on 28 November
2024) was used to further confirm Tophree 50 ASVs and other differentially enriched
microorganisms, reclassifying them as needed.
4.4. Ileal Morphology
A hematoxylin and eosin (H&E) staining technique was routinely applied to ileum
samples that were obtained midway between Meckel’s diverticulum and the ileocecal
junction. The samples were cut into 5 µm thick sections and embedded in paraffin. Pho-
tomicrographs were taken using a camera ICC50W associated with a microscope Leica
DM2500 (Leica, Wetzlar, Germany). The variables measured were the villus height, villus
width, and the villus cross-sectional area (villus height × villus width). Morphometric
measurements were performed using the ImageJ 1.52v software. Every variable in each
treatment considered 63 measurements (9 per replicate).
4.5. Barrier Function
Intestinal permeability was assessed by measuring the levels of the biomarker fluores-
cein isothiocyanate dextran (FITC-d, molecular weight 3–5 kDa, Merck KGaA, Darmstadt,
Germany) in the serum. To achieve this, FITC-d (8.32 mg/kg) was given orally to twenty-
one poults per treatment one hour before euthanasia. The serum samples were processed
according to the recommendations of Baxter et al. [39], and fluorescence measurements
were performed at 485 nm excitation and 528 nm emission using a Synergy HT multimode
micro plate reader (Bio Tek Instruments, Inc., Winooski, VT, USA).
4.6. Immunity
The skin response to phytohemagglutinin m (PHA-M) was used to measure cellular im-
mune activity using cutaneous basophil hypersensitivity (CBH). On day 28, three poults per
replicate were randomly selected and injected intradermally in the interdigital skin between
the third and fourth digits of the left foot with 0.1 mL of PHA m (Gibco, Grand Island, NY,
USA). The CBH response was calculated by using the following mathematical expression:
CBH (mm) = (thickness 24 h postinjection)− (thickness preinjection)
4.7. Statistical Analysis
The ASV tables were standardised through the application of cumulative sum scaling
(CSS) within the R (v. 1.4.0) metagenome Seq package [40]. The phyloseq package v.
1.42.0 was used to calculate the α-diversity (Shannon’s Index, Observed ASV, and Pielou’s
Evenness) and β-diversity (unweighted and weighted UniFrac distances), which were
then plotted using the ggplot2 program in R [41]. The statistical significance of α-diversity
and relative abundance was assessed by a non-parametric Kruskal–Wallis test, which
was followed by a pairwise Wilcoxon rank sum test. Using the ADONIS function in the
vegan package v. 2.6.4, the non-parametric permutational multivariate analysis of variance
(PERMANOVA) was used to determine the significance in β-diversity [42]. To determine
the differential enrichment of bacteria ASVs among different groups of bacteria, linear
Int. J. Mol. Sci. 2024, 25, 7977 11 of 13
discriminant analysis (LDA) effect size (LEfSe) was employed, with a threshold of p < 0.05
and an LDA score of ≥3.0 [43]. Finally, morphometric, serum concentrations of FITC-d,
and CBH data were all subjected to ANOVA in a completely randomised design, utilising
the general linear model process in SAS [44]. By using the Tukey multiple range test at
p < 0.05, means were separated.
4.8. Data Availability
Raw sequencing reads were deposited in the NCBI GenBank SRA database under the
accession number PRJNA1020155.
5. Conclusions
In the present study, the results indicated that the addition of alfalfa adsorbent pro-
moted the proliferation of beneficial bacteria, like Faecalibacterium and Coprococcus catus,
increased the height of the villi and significantly enhanced intestinal permeability, which
was in contrast to the AF group. Finally, we evaluated the complex link between dietary
supplementation with alfalfa, AFB1, and the cellular immune response. The significant
increase in CBH in response to phytohemagglutinin injection in turkey poults supple-
mented with alfalfa and AFB1 may be explained by the fact that alfalfa and AFB1 interact to
boost the immunological response, since alfalfa contains components that may counteract
the immunosuppressive effects of AFB1, resulting in a more robust immune response. In
conclusion, the results of this study indicate that alfalfa may serve as an effective dietary
intervention for the mitigation of the adverse effects of AFB1 in turkey poults.
Author Contributions: Conceptualization, J.A.M.-G., J.L., S.G.-R., A.M.-A. and G.T.-I.; methodol-
ogy, M.d.J.N.-R., J.O.H.-R., J.D.L. and R.S.-C.; software, M.d.J.N.-R. and A.V.-D.; validation, G.Z.,
S.G.-R., A.M.-A. and G.T.-I.; formal analysis, M.d.J.N.-R. and A.V.-D.; investigation, J.D.L., A.S. and
R.S.-C.; resources, B.M.H. and G.T.-I.; data curation, M.d.J.N.-R. and J.L.; writing—original draft
preparation, J.A.M.-G., J.L., S.G.-R. and A.M.-A.; writing—review and editing, X.H.-V. and G.T.-I.;
visualization, J.L. and J.A.M.-G.; supervision, S.G.-R., A.M.-A. and G.T.-I.; project administration and
funding acquisition, B.M.H. and G.T.-I. All authors have read and agreed to the published version of
the manuscript.
Funding: This project was funded by UNAM-PAPIIT grant number IA101523 and by funds pro-
vided by USDA Animal Health Awards (FY2021 & FY2022), as well as by USDA-NIFA Sustain-
able Agriculture Systems, Grant No. 2019-69012-29905. Title of Project: Empowering U.S. Broiler
Production for Transformation and Sustainability USDA-NIFA (Sustainable Agriculture Systems):
No. 2019-69012-29905.
Institutional Review Board Statement: All animal handling procedures complied with the Institu-
tional Animal Care and Use Committee (IACUC) at the University of Arkansas, Fayetteville (protocol
No. 22020).
Informed Consent Statement: Not applicable.
Data Availability Statement: Upon reasonable request, and subject to review, the authors will
provide the data that support the findings of this study.
Conflicts of Interest: The authors declare no conflicts of interest.
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51 
 
Discusión  
 
Experimentos de adsorción in vitro 
 
En la presente investigación, se evaluó la capacidad adsorbente de AFB1 de un adsorbente orgánico 
derivado de hojas de alfalfa usando dos modelos in vitro (un modelo pH-dependiente y un modelo 
que emuló el TGI de las aves). Varios métodos in vitro han proporcionado una mayor comprensión 
en cuanto a la afinidad y la capacidad de unión entre los agentes adsorbentes y las micotoxinas, lo 
que los convierte en herramientas útiles para determinar su eficacia de adsorción (Diaz et al., 2003). 
La mayoría de los estudios con modelos in vitro, se han enfocado en utilizar soluciones buffer con 
pHs que van desde 3 a 7, principalmente, con el fin de imitar el pH estomacal e intestinal de los 
animales, respectivamente (Kolawole et al., 2019).  
En este estudio, se observó que el uso del adsorbente de hojas de alfalfa (0.5%, p/v) en un modelo 
in vitro pH-dependiente con tres diferentes valores de pH (2, 5 y 7), y a una concentración de 250 
ng de AFB1/mL, no mostró una diferencia estadísticamente significativa en el porcentaje de 
adsorción de AFB1 en ninguno de los tres valores de pH (2, 5 y 7), dando porcentajes del 98.2%, 
99.9% y 98.2%, respectivamente. Se ha comprobado que el pH de una solución puede afectar 
significativamente la capacidad de adsorción de un adsorbato en un medio acuoso debido a las 
interacciones electrostáticas existentes entre la carga superficial del adsorbente y el adsorbato 
(Abollino et al., 2003). Si la interacción electrostática fuera el único mecanismo para la adsorción de 
AFB1, entonces la capacidad de eliminación debería ser máxima dentro del rango de pH 2-5 (pHpcz = 
6.51). En este rango de pH, la superficie del adsorbente de hojas de alfalfa está cargada 
positivamente y los átomos de oxígeno de la molécula de AFB1 están cargados negativamente, lo 
cual permitiría la formación de enlaces de hidrógeno entre el adsorbente y el adsorbato (Al-Degs et 
al., 2008). Sin embargo, el pH en el presente estudio no ejerció ninguna influencia en la eliminación 
de AFB1 en este modelo in vitro, lo que sugiere que podría estar involucrada algún otro tipo de 
interacción. 
Sin embargo, estas condiciones no reflejan completamente el entorno del TGI de los animales, dado 
que otros factores como la temperatura, el tiempo de paso del alimento, la presencia de enzimas 
digestivas, el tipo de alimento, las sales biliares y los nutrientes pueden influir en el proceso de 
adsorción, por lo que, algunos investigadores han desarrollado modelos in vitro que emulen con 
52 
 
mayor precisión el TGI de los animales (Di Gregorio et al., 2014; Kolawole et al., 2019; Vázquez-
Durán et al., 2022). 
En este estudio, también se empleó un modelo que emula el TGI de las aves, en este modelo se 
observó que el uso del adsorbente de hojas de alfalfa (0.5%, p/v), a una concentración de 250 ng de 
AFB1/mL disminuyó su porcentaje de adsorción (88.8%). Esto se atribuyó a la dificultad que encontró 
el adsorbente para capturar la molécula AFB1 en un entorno más complejo que contiene pepsina y 
sales biliares (Li et al., 2016). Además, Oliveira et al. (2019) reportaron que la pérdida y generación 
o liberación de compuestos es bastante común durante la digestión, principalmente en la sección 
intestinal, debido a la actividad de las sales biliares. Además, el impedimento estérico en moléculas 
orgánicas puede obstaculizar o limitar la proximidad entre las moléculas en una reacción química, 
afectando así la capacidad de adsorción de las micotoxinas (Avantaggiato et al., 2014). Finalmente, 
según Avantaggiato et al. (2004), la presencia de diversas sustancias, como coccidiostáticos, 
vitaminas, minerales, aminoácidos y otros componentes de la dieta, podrían influir en la capacidad 
del adsorbente para unirse a niveles bajos de AFB1. En ambos modelos in vitro, el porcentaje de 
adsorción de AFB1 de la PCL fue significativamente menor, en comparación con el adsorbente 
orgánico.  
 
Caracterización del adsorbente de hojas de alfalfa 
 
La caracterización de los adsorbentes orgánicos de micotoxinas se lleva a cabo mediante diversas 
técnicas con el objetivo de comprender los principales mecanismos físicos y químicos implicados en 
la adsorción (Vázquez-Durán et al., 2022). 
La espectroscopia FTIR es una técnica ampliamente empleada para caracterizar a los adsorbentes 
orgánicos de micotoxinas debido a que proporciona información detallada de la presencia de los 
grupos funcionales de los materiales (Pellenz et al., 2023). Los adsorbentes orgánicos están 
compuestos principalmente por proteínas, carbohidratos, lípidos, curcuminoides, flavonoides, 
alcaloides, esteroides, terpenoides, saponinas, fenólicos, glucósidos y clorofilas. Todos estos 
componentes presentan diversos grupos funcionales, como hidroxilo, amino, carboxilo, carboxilato, 
amida, fosfato, éster y cetona, que pueden desempeñar un papel parcial en la adsorción de 
aflatoxinas (Adunphatcharaphon et al., 2020; Karmanov et al., 2020; Shar et al., 2016). El material 
adsorbente que se estudió en esta investigación, presentó principalmente cinco grupos funcionales: 
los hidroxilo, las cadenas alifáticas, los compuestos aromáticos, los carboxilos y los carbonilos. Se ha 
53 
 
documentado que la adsorción de AFB1 puede ser facilitada por la hidrofobicidad de los 
adsorbentes, la cual se atribuye a la presencia de grupos hidrofóbicos como los metilos y los grupos 
aromáticos en su superficie (Vázquez-Durán et al., 2021). 
Teniendo en cuenta que el pH del medio podría influir en la adsorción de AFB1, es crucial determinar 
la constante de disociación ácida (pka) de los grupos funcionales presentes en el adsorbente de 
hojas de alfalfa. El pka indica la tendencia de un ácido a ceder un protón (H⁺) en una solución. Los 
grupos funcionales se desprotonan cuando el pH del medio supera su pKa (Nogueira et al., 2022). 
Se sabe que un menor valor de pKa indica una mayor fuerza ácida, lo que implica una mayor 
tendencia para perder o liberar un protón en una solución acuosa. Por el contrario, un mayor valor 
de pKa señala un ácido más débil y menos propenso a ceder un protón. Esencialmente, determinar 
el pKa proporciona información valiosa sobre la capacidad del adsorbente para interactuar con AFB1 
en diferentes condiciones de pH (Nogueira et al., 2022). Dado que el pKa del grupo hidroxilo es de 
~11.6, del grupo amina es de ~40 y del grupo amida es de ~18, a un pH más bajo, aproximadamente 
por debajo de ~11.6, ~40 y ~18 para el grupo hidroxilo, la amina y la amida respectivamente, estarán 
principalmente protonados, mientras que, a un pH más alto, estarán principalmente desprotonados. 
Esta condición facilita la formación de enlaces de hidrógeno con los átomos de oxígeno de la 
molécula de AFB1 en las tres secciones simuladas del TGI (pH 2, 5 y 7) de las aves (Figura 1). La 
presencia del grupo carboxilo solo permite la formación de enlaces de hidrógeno a pH de 2 debido 
a que su pka es de ~4.5. Es relevante destacar que el pKa de la molécula AFB1 es de 17.79, lo que 
indica que en el intervalo de pH 2 a 7, la AFB1 no se encuentra ni protonada ni desprotonada. Por lo 
tanto, en el contexto de este estudio, el pH no afectó la adsorción de AFB1, independientemente de 
la carga superficial del adsorbente. 
 
 
 
 
 
 
 
 
54 
 
Figura 1. Formación de enlaces de hidrógeno entre la molécula de AFB1 y los principales grupos funcionales 
existentes en el adsorbente de alfalfa en las tres secciones simuladas del tracto gastrointestinal de las aves 
(pH 2, 5 y 7). 
 
La microscopía electrónica de barrido (MEB) destaca como una de las técnicas más empleadas para 
el análisis detallado de la microestructura y morfología. Es ampliamente reconocido que las 
propiedades de adsorción de los adsorbentes orgánicos pueden estar vinculadas tanto a sus 
características estructurales como químicas ya que las cavidades proporcionan un área de unión 
entre la superficie del adsorbente y el adsorbato (Malik et al., 2015). Por lo general, la capacidad de 
adsorción de aflatoxinas aumenta proporcionalmente con la cantidad de poros o cavidades 
presentes en la superficie de los biomateriales (Vázquez-Durán et al., 2022). La espectroscopia de 
fluorescencia de rayos X (EFRX) constituye una técnica de microanálisis químico típicamente 
complementaria al MEB, ya que se aplica extensamente para la identificación elemental y el mapeo 
compositivo de muestras sólidas (Hodoroaba, 2020; Shindo et al., 2002). El adsorbente derivado de 
hojas de alfalfa, exhibe una estructura caracterizada por una superficie rugosa con algunos bordes 
dispuestos en una configuración laminar. Estos espacios entre las estructuras desordenadas y 
heterogéneas podrían proporcionar una amplia disponibilidad de sitios de superficie y una mayor 
exposición de los grupos funcionales presentes en el adsorbente de hojas de alfalfa. Esta disposición 
55 
 
favorece la adsorción de AFB1, los resultados concuerdan con lo reportado previamente por Shar et 
al. (2016) quienes observaron una mejor adsorción de aflatoxinas con el adsorbente elaborado a 
partir de cáscaras de plátano secadas en horno, el cual exhibía una estructura superficial rugosa y 
un tamaño de poro más grande en comparación con el adsorbente elaborado a partir de cáscaras 
de plátano liofilizadas (Wang et al., 2020). En cuanto al análisis elemental, el adsorbente preparado 
a partir de hojas de alfalfa mostró una cantidad significativa de tres elementos principalmente: 
carbono (49.44%), oxígeno (43.92%) y nitrógeno (5.48%), lo cual resulta esencial para el proceso de 
eliminación de la AFB1, estos resultados son similares a los reportados por Vázquez-Durán et al. 
(2023) quienes observaron que el adsorbente a base de cuscuta, presentó valores de 51.24% de 
carbono, 43.52% de oxígeno y 4.62% de nitrógeno, los cuales son esenciales en la capacidad 
adsorbente de contaminantes.  
La técnica de difracción de rayos X (DRX) es una técnica que se usa para investigar las propiedades 
cristalinas de los materiales. Esta técnica permite evaluar la disposición atómica, el tamaño de los 
cristales, los parámetros de la red y las imperfecciones en los materiales estudiados (Pellenz et al., 
2023). Los patrones de DRX para el adsorbente derivado de las hojas de alfalfa mostró una 
estructura amorfa distintiva basada en amplios picos de difracción, presentando tres picos de 
intensidad considerable a 2θ = 25.04°,13.72° y 17.02°, los cuales se asociaron con la presencia de 
celulosa cristalina y almidón semicristalino. Diversos autores han señalado que los desechos 
agrícolas se caracterizan por un alto contenido de celulosa, hemicelulosa, lignina, almidón y ceniza 
los cuales contienen una gran variedad de grupos funcionales formados principalmente por carbono 
y oxígeno, lo que les confiere propiedades favorables para la adsorción de contaminantes y metales 
pesados (Hashem et al., 2006; Sud et al., 2008). En contraste con los resultados obtenidos del 
espectro FTIR, se observó una banda entre los 3668 y 3280 cm−1, la cual corresponde a las 
vibraciones de estiramiento de O-H y los enlaces H asociados a la celulosa, pectina y lignina, los 
principales componentes de adsorción según lo reportado por Adunphatcharaphon et al. (2020). La 
celulosa, al ser un homopolímero cristalino de glucosa con enlaces de hidrógeno intramoleculares e 
intermoleculares (Sud et al., 2008), tendría la capacidad de formar una gran cantidad de enlaces de 
hidrógeno con los oxígenos de la molécula de AFB1. 
Por otro lado, el punto de carga cero (pHpzc) ofrece información relevante sobre la carga neta 
superficial de los adsorbentes orgánicos y se define como el pH en el cual la carga superficial del 
adsorbente se equilibra y alcanza cantidades iguales en la capacidad de intercambio catiónico y 
aniónico. Se ha sugerido que cuando el pH es menor que el pHpzc, la superficie del biomaterial 
56 
 
adquiere una carga positiva, mientras que, si el pH es mayor que el pHpzc, la superficie estará cargada 
negativamente (Lim et al., 2017; Zavala-Franco et al., 2018). 
Se determinó que el pHpzc del adsorbente derivado de las hojas de alfalfa fue de 6.51. En 
consecuencia, la superficie del adsorbente está cargada positivamente para valores de pH inferiores 
a 6.51 y negativamente para valores de pH superiores a éste, por lo que en el rango de pH de 2 a 5, 
la interacción entre la superficie del adsorbente de hojas de alfalfa y los átomos de oxígeno 
(cargados negativamente) de la molécula de AFB1 permitirían la interacción electrostática entre el 
adsorbente y el adsorbato (Figura 2). Sin embargo, en el experimento in vitro dependiente del pH, 
no se observaron diferencias estadísticamente significativas en la adsorción de AFB1, lo que sugiere 
que, a un pH de 7, la interacción electrostática no constituye el principal mecanismo de adsorción, 
lo cual concuerda con lo reportado por Adunphatcharaphon et al. (2020).  
 
Figura 2. Interacción electrostática de acuerdo con el pHpzc entre la superficie del adsorbente de hojas de 
alfalfa y los átomos de oxígeno de la molécula de AFB1. 
 
 
 
 
 
57 
 
El potencial ζ también se emplea para estudiar la carga superficial de los adsorbentes y sus posibles 
interacciones con el adsorbato y se determina experimentalmente a través de la medición de la 
movilidad electroforética de las partículas sólidas dispersas en un líquido al aplicar un campo 
eléctrico en la suspensión (Pellenz et al., 2023).  
El potencial ζ del adsorbente derivado de las hojas de alfalfa fue positivo a pH 2 y significativamente 
más negativo en los pHs de 5 y 7. Por lo tanto, se propone que existe una contribución significativa 
a la adsorción de AFB1 cuando se utiliza el adsorbente en pH ácidos (pH 2), debido a las interacciones 
electrostáticas entre el adsorbente y la AFB1 (Figura 3). Las interacciones electrostáticas varían 
según el pH de la solución; por ende, en este estudio, las atracciones iónicas tienen una influencia 
menor en el proceso de adsorción de AFB1.  
El punto isoeléctrico (pI) se define como el pH en el cual una molécula o una superficie sólida lleva 
una carga neta igual a cero, debido al equilibrio entre los grupos funcionales que pueden ionizarse 
en la superficie. Cuando el pH de una solución es menor que el pI, predominarán las cargas positivas 
en la estructura. Esto ocurre debido al exceso de iones H+ en la solución, lo que provoca que los 
grupos funcionales ácidos (como los grupos carboxilo) presentes en el adsorbente estén 
predominantemente protonados. El pI del adsorbente de hojas de alfalfa fue de 2.2, por lo que se 
esperaría que a un pH inferior a su pI, la superficie del adsorbente interactúe con la molécula de 
AFB1 mediante la formación de enlaces de hidrógeno con los grupos oxígeno de la AFB1, dando 
mejores resultados de adsorción (Figura 3). 
 
 
 
 
 
 
 
 
 
 
 
 
 
58 
 
Figura 3. Interacción en la interfase del potencia ζ y el punto isoeléctrico (pI) entre el adsorbente de hojas de 
alfalfa y los átomos de oxígeno de la molécula de AFB1. 
 
 
 
Varios estudios han demostrado que la clorofila, es capaz de formar complejos no covalentes con la 
AFB1, independientemente de la temperatura o el pH (Arimoto et al., 1993; Nava-Ramírez et al., 
2021; Simonich et al., 2007). Por lo tanto, en el presente estudio se realizó la determinación 
cuantitativa de la clorofila y los carotenoides mediante las técnicas de espectroscopía de UV-Vis y la 
espectroscopía de fluorescencia, las cuales son útiles para evaluar el contenido de los pigmentos 
fotosintéticos de los adsorbentes orgánicos. Para determinar el contenido preciso de los principales 
pigmentos fotosintéticos en el adsorbente de alfalfa, se emplearon los coeficientes de absorción 
específicos desarrollados por Lichtenthaler y Buschmann. (2001), utilizando la técnica de 
espectroscopía de UV-Vis. Se observó que el contenido total de clorofila en el adsorbente preparado 
a partir de hojas de alfalfa, fue significativamente mayor que el contenido total de carotenoides. El 
espectro de fluorescencia del adsorbente de alfalfa muestra dos máximos de fluorescencia (690 nm 
y 735 nm), los cuales corresponden a la fluorescencia de la clorofila. Por consiguiente, se demostró 
que la alfalfa posee una cantidad considerable de clorofilas totales.  
Recientemente, nuestro grupo de investigación llevaron a cabo un estudio teórico y una serie de 
experimentos in vitro en donde se demostró la posible formación de complejos entre la clorofila y 
59 
 
la AFB1. En el estudio teórico (Vázquez-Durán et al., 2022), se sugiere que la formación de complejos 
clorofila-AFB1 se debe principalmente a la interacción entre los cationes Mg2+ de la clorofila y los 
átomos de oxígeno del sistema β-dicarbonilo de la molécula de AFB1 (Figura 4). Sin embargo, 
también podría existir la formación de enlaces de hidrógeno entre: a) los iones H+ de la molécula de 
AFB1 y los oxígenos de los grupos éster carbonilo, carbonílico del acetato de fitol y cetónico de la 
molécula de clorofila y b) el oxígeno del grupo furano de la AFB1 y los H+ de los grupos cetónicos y 
éster de la clorofila.  
 
Figura 4. Interacción entre la clorofila del adsorbente de las hojas de alfalfa y la molécula de la AFB1. 
 
 
Finalmente, se ha reportado que, la clorofila, una molécula altamente hidrofóbica, puede formar 
complejos no covalentes con la molécula AFB1, independientemente del pH. En los experimentos 
previos in vitro se utilizaron adsorbentes a base de lechuga, cola de caballo (Nava-Ramírez et al., 
2020) y kale (Vázquez-Durán et al., 2021). En el primer experimento, se emplearon adsorbentes de 
lechuga y cola de caballo en un modelo in vitro acuoso pH-dependiente y se observaron tasas de 
adsorción máximas de AFB1 del 95% para la lechuga y del 71% para la cola de caballo. El segundo 
experimento fue realizado con un modelo in vitro que emuló el TGI de las aves, en este estudio se 
mostraron porcentajes de adsorción de AFB1 del 94% para el kale y del 84% para la lechuga. Ambos 
60 
 
estudios demostraron que los adsorbentes que contenían mayores cantidades de clorofilas 
exhibieron una mayor capacidad de adsorción de AFB1. Estos resultados concuerdan con el presente 
estudio. La combinación de estas interacciones, tanto electrostáticas como no electrostáticas, 
confiere al adsorbente derivado de las hojas de alfalfa una eficacia notable en la adsorción de AFB1 
presente en el alimento contaminado destinado a las aves. 
 
Eficacia del adsorbente de hojas de alfalfa en pavitos 
 
Considerando que los modelos in vitro podrían no replicar plenamente las condiciones fisiológicas 
del TGI de las aves, lo cual puede influir en la predicción de los resultados, se optó por evaluar la 
eficacia del adsorbente de hojas de alfalfa, agregado en una dieta contaminada con 250 ng de AFB1/g 
de alimento, destinada a pavitos Nicholas-700 de un día de edad. Se utilizó este modelo ya que los 
pavos tienen una capacidad limitada para metabolizar y detoxificar la AFB1. Sus sistemas 
enzimáticos, como el citocromo P450, que están involucrados en la destoxificación de toxinas, no 
son tan eficientes como en otras aves. 
En la semana final del experimento (28 días de edad), se observó que el grupo de AF tuvo una 
reducción significativa en el peso corporal, la ganancia de peso y el consumo de alimento, en 
comparación con el grupo control. Sin embargo, cuando se agregó el adsorbente de hojas de alfalfa, 
los parámetros productivos de los pavitos mejoraron significativamente. Guerrini y Tedesco. (2023) 
observaron resultados similares a los del presente estudio al agregar cardo mariano en una dieta 
contaminada con AFB1 destinada para pollos de engorda, observando una mejoría en los parámetros 
productivos. Los autores atribuyen que el tratamiento es eficaz para contrarrestar los efectos 
tóxicos de la AFB1 debido a los efectos hepatoprotectores, antioxidantes, y antiinflamatorias del 
cardo mariano. 
Además, Adams et al. (2019) observaron que la suplementación de fibra dietética de alfalfa en la 
dieta de lechones promovió el crecimiento y la salud de los lechones. Los componentes de la fibra 
pueden ser fermentados por microorganismos en el TGI, generando grandes cantidades de ácidos 
grasos de cadena corta, que sirven como principal fuente de energía y para la reducción de la 
población de bacterias entéricas en el intestino. Es importante recalcar que el grupo de Alfalfa fue 
el que obtuvo los mejores valores de los parámetros productivos en los pavitos. Oyegunwa et al. 
(2021) informaron que ofrecer una dieta experimental contaminada con 150 ng de AFB1/g de 
alimento durante un período de 28 días, produjo una disminución significativa en el peso corporal, 
61 
 
la ganancia de peso y el consumo de alimento de los pavitos. Algunos autores han demostrado que 
la eficacia de los subproductos de la uva en la mejora de los parámetros productivos se atribuye 
principalmente a la influencia de sus biomoléculas activas en la composición microbiana del 
intestino y a la regulación negativa de varios genes proinflamatorios en diferentes regiones 
intestinales (Taranu et al., 2019). 
Este es el primer experimento en pavitos en donde se estudió el efecto adsorbente de AFB1 de las 
hojas de alfalfa; sin embargo, se han realizado algunos experimentos para reducir los efectos tóxicos 
causados por la AF en aves utilizando productos y subproductos de origen vegetal con propiedades 
antioxidantes, antiinflamatorias y hepatoprotectoras como Satureja khuzistanica, Zataria multiflora 
Boiss, Thymus vulgaris, Sauropsus androgynus, Hemidesmus indicus, Leucas aspera, Moringa 
oleifera, Eclipta alba, Curcuma longa, Silybum marianum, Urtica dioica, and cítricos (Umaya et al., 
2021).  
En cuanto al peso relativo de los órganos, en contraste con el grupo de control, se observó una 
disminución significativa en el peso relativo del hígado en los pavitos de los otros cuatro grupos 
experimentales (Alfalfa, AF, AF+alfalfa y AF+PCL). El peso relativo del bazo y de la bolsa de Fabricio 
no tuvo diferencias estadísticas significativas entre los grupos que fueron alimentados con AFB1 (AF, 
AF+alfalfa y AF+PCL). Por otro lado, el grupo de Alfalfa no presentó diferencias estadísticamente 
significativas en comparación con el grupo control. Estos hallazgos son consistentes con los 
informados por Sridhar et al. (2015) quienes afirmaron que el aumento en el peso relativo de ciertos 
órganos producido por el consumo de aflatoxinas se revirtió exitosamente gracias a la 
suplementación con resveratrol en la dieta de pollos de engorde. 
Es posible que las alteraciones orgánicas inducidas por la AFB1 se hayan atenuado gracias al 
adsorbente orgánico, que impidió la disociación de la molécula de AFB1 a lo largo del TGI de las aves. 
Esto podría haber evitado su metabolización en el órgano blanco, el hígado. Además, estos agentes 
orgánicos parecen disminuir la inflamación, la necrosis, y la fibrosis, al mismo tiempo que 
intervienen en el metabolismo de los lípidos hepáticos (Lakkawar et al., 2004). 
Se ha reportado que algunos materiales naturales mitigan la toxicidad inducida por la AFB1 mediante 
diversos mecanismos, incluyendo la inhibición del estrés oxidativo, la supresión de la respuesta 
inflamatoria, la inhibición de la apoptosis y la modulación de la respuesta inmune (Dai et al., 2022).  
Estos materiales son ricos en saponinas, polifenoles, ácidos grasos poliinsaturados, vitaminas y 
minerales los cuales tienen una fuerte actividad antioxidante, lo que podría aumentar la capacidad 
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de defensa del animal frente a la acción negativa de las micotoxinas, aumentando la absorción de 
antígenos del intestino (Pietrzak y Grela, 2015; Taranu et al., 2019). 
En cuanto a la bioquímica sanguínea, se observó una reducción en los valores de PT, Glu, Ca y Cr en 
los grupos AF, AF+alfalfa y AF+PCL en comparación con el grupo control. Sin embargo, el grupo de 
Alfalfa no mostró diferencias estadísticamente significativas en comparación con el grupo control, 
los cuales presentaron un aumento significativo en los niveles de PT, Glu, Ca y Cr en relación con los 
grupos alimentados con AFB1. Se ha informado que las aflatoxinas causan una disminución 
significativa en el contenido de creatinina (Gómez-Espinosa et al., 2017), glucosa (Sridhar et al., 
2015) y calcio (Gowda et al., 2009) en aves de corral. Esto está estrechamente relacionado con lo 
observado en el estudio actual. Por último, el ácido úrico no mostró diferencias significativas entre 
los cinco grupos de pavitos, estos hallazgos concuerdan estrechamente con los resultados 
encontrados por Quist et al. (2000). Pietrzak y Grela. (2015) reportaron un mayor contenido de 
proteínas totales en el plasma sanguíneo, sin observan cambios significativos en los niveles de ácido 
úrico, esto lo atribuyen al efecto positivo de la suplementación con un concentrado de proteínas de 
alfalfa en el metabolismo de las proteínas en el animal. 
En general, un incremento en la actividad de las enzimas hepáticas se asocia comúnmente con 
degeneración hepatocelular; por lo tanto, se produce una fuga de estas enzimas a la circulación 
sanguínea (Singh, 2019). En cuanto a la concentración de las enzimas hepáticas ALP, AST, CK y GLDH, 
no se encontraron diferencias estadísticas significativas entre los cinco tratamientos. La ALT fue la 
única enzima en la que se observó un marcado aumento en el grupo AF+PCL. Quist et al. (2000) no 
encontraron diferencias significativas en el nivel de AST entre pavos que consumieron una dieta 
contaminada con 200 ng de AFB1/g de alimento y el grupo control. Además, Gómez-Espinosa et al. 
(2017) no observaron diferencias significativas en los niveles de ALT y ALP en pavitos alimentados 
con 430 ng de AFB1/g de alimento en comparación con el grupo de control. Algunos autores han 
demostrado los efectos positivos en los niveles de enzimas hepáticas debido a la adición de algunos 
materiales orgánicos. Fani et al. (2013) informaron que la adición de semillas de Silybum marianum 
(SMS) en una dieta de pollo de engorde contaminada con 250 ng AFB1/g, no produjo diferencias 
significativas en los niveles de AST y ALT en comparación con el grupo control. Los autores 
concluyeron que los SMS podrían prevenir los efectos adversos causados por la AFB1 en las aves, 
debido a su efecto antioxidante y antiinflamatorio. En línea con estos resultados, Masouri et al. 
(2022) demostraron que la adición de menta en polvo a una dieta contaminada con aflatoxinas 
destinada a codornices japonesas alivió los efectos causados por las aflatoxinas, sin encontrar 
63 
 
diferencias significativas en los niveles de ALT y AST en comparación con el grupo de control. El alivio 
de los efectos negativos de la toxina se debe probablemente al impedimento del adsorbente en la 
presentación de estrés oxidativo en los hepatocitos y a la prevención de la inflamación hepática 
(Taranu et al., 2020).  
Finalmente, en cuanto a la histología del hígado, en el grupo de AF se observó un incremento 
significativo en la severidad de las lesiones hepáticas, evidenciando características como 
degeneración vacuolar, inflamación, hiperplasia de los conductos biliares y fibrosis. Sin embargo, en 
los dos grupos experimentales suplementados con hojas de alfalfa, no se registraron diferencias 
significativas en las lesiones hepáticas en comparación con el grupo de control. Estos hallazgos 
concuerdan con lo reportado por Perali et al. (2020) quienes señalaron que los efectos negativos de 
1018 μg de AFB1/kg de alimento sobre el peso relativo, los cambios macroscópicos y microscópicos 
en el hígado y los parámetros bioquímicos mejoraron con la adición de 0.2% de Lithothamnium 
calcareum debido a su efecto adsorbente de AFB1 aunado a su aporte nutricional sobre las aves. 
Wang et al. (2022) demostraron que la adición de la curcumina redujo la acumulación de aductos 
de ADN-AFB1 en el hígado, aliviando la hepatotoxicidad al inhibir el estrés oxidativo inducido por 
AFB1 y potenciar la desintoxicación mediada por las enzimas de conjugación de la fase II, a través de 
la acción de la GST. Asimismo, inhibió la respuesta inflamatoria inducida por AFB1 y el estrés 
oxidativo mediante la regulación positiva del factor nuclear eritroide 2 (Nrf2).  
Adicionalmente, se estudió el efecto del adsorbente de alfalfa sobre la microbiota intestinal, la 
permeabilidad intestinal, el análisis de la morfología del íleon y la inmunidad celular en los pavitos 
a los 28 días de edad. En general, los cinco grupos de aves no presentaron diferencias estadísticas 
significativas en cuanto a la diversidad α y β de la microbiota cecal, a excepción de la diversidad β, 
según la distancia no ponderada de UniFrac, la cual indica una diferencia estadística significativa 
entre el grupo Control y el grupo AF. En este contexto, Maguey-González et al. (2023) reportaron 
resultados similares al agregar ácidos húmicos (AH) en una dieta contaminada con AFB1 destinada 
para los pavos. Los autores encontraron una diferencia estadísticamente significativa en la 
diversidad β, entre el grupo control y el grupo que consumió la dieta contaminada con AFB1, lo que 
indicó la influencia de AFB1 en la composición de la microbiota intestinal. Sin embargo, no se 
encontró diferencia estadísticamente significativa entre el grupo control y el grupo de pavos que 
consumieron los AH, lo que sugiere una mejoría en la restauración de la microbiota intestinal y un 
aumento en la presencia de bacterias benéficas. 
64 
 
En cuanto a las abundancias relativas (%) de bacterias cecales, la familia Streptococcaceae mostró 
una disminución en su población en el grupo Control en comparación con los cuatro grupos 
restantes. Además, la abundancia relativa de los géneros de Coprobacillaceae se redujo 1.19 y 4.41 
veces en los grupos AF y Alfalfa+AF, respectivamente, en comparación con el grupo Control. 
Asimismo, la abundancia relativa de las variantes de secuencia de amplicones (ASV) de 
Coprobacillaceae se redujo 3.91 y 2.49 veces en los grupos AF y Alfalfa+AF, respectivamente, en 
comparación con el grupo Control. En contraste, el grupo Alfalfa mostró un aumento significativo 
en los niveles de Faecalibacterium en comparación con el resto de los grupos experimentales. La 
presencia de la familia Streptococcaceae está asociada con mejores parámetros productivos en las 
aves, por lo que, su presencia, se asocia comunmente a una microbiota intestinal saludable 
(Marková et al., 2024). Por otro lado, la familia Coprobacillaceae se encuentra en la mayoría de los 
conjuntos de datos de microbiomas de diversos huéspedes. Sin embargo, no está bien estudiada y 
hay poca información disponible (Liu et al., 2015). Según Rychlik, (2020), el género Faecalibacterium 
tiene la capacidad de consumir oxígeno a bajas concentraciones, lo que mantiene un ambiente 
estrictamente anaeróbico. Esto restringe la capacidad de Escherichia coli y Salmonella para utilizar 
el metabolismo respiratorio anaeróbico, evitando así su crecimiento excesivo. 
En el enriquecimiento diferencial de ASV bacteriano, se encontró una mayor abundancia de 
Mediterraneibaer (F91) y Lachnospiracaeae (F25) en el grupo Contro; Faecalibacterium (F7 y F53) y 
Coprococcus catus (F85) en el grupo Alfalfa; Bacillus (F8) y Anaerotignum (F99) en el grupo 
AF+Alfalfa; mientras que el grupo AF tuvo mayores abundancias de Streptococcus lutetiensis (F6).  
De acuerdo con lo reportado por Monson et al. (2015), el consumo de una dieta contaminada con 
AFB1, incluso en contenidos bajos, puede dañar el sistema inmunológico de las aves, el cual depende 
de la bolsa de Fabricio, el timo, y el bazo. Por lo tanto, la ingesta de AFB1 durante el crecimiento 
puede provocar atrofia del tejido inmunológico y suprimir las respuestas inmunes innatas y 
adaptativas, lo que puede llevar al aumento del crecimiento de bacterias patógenas como 
Streptococcus lutetiensis (Maguey-González et al., 2024). Por otra parte, se ha informado que la 
abundancia de Coprococcus catus y de Faecalibacterium, bacterias productoras de butirato, son 
potencialmente benéficas para el intestino.  Además, se ha observado que Faecalibacterium puede 
disminuir la inflamación y prevenir el aumento de la permeabilidad intestinal (Richards et al., 2019; 
Yang et al., 2023). De esta manera, es posible asociar el consumo del adsorbente de alfalfa con una 
mejor microbiota intestinal (Figura 5).  
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Por otra parte, en el análisis morfométrico del íleon, se observó que los grupos Control y Alfalfa 
presentaron una mayor altura de las vellosidades, seguido de los grupos AF+PCL y AF+alfalfa. El 
grupo AF mostró la menor altura de las vellosidades, siendo 2.12 veces menor en comparación con 
el grupo Control. Además, el grupo Control presentó un área total de las vellosidades mayor en 
comparación con los cuatro grupos restantes. Sin embargo, el grupo AF presentó la menor área total 
de las vellosidades, siendo 2.31 veces menor en comparación con el Control. Estos resultados 
concuerdan con lo reportado por Rajput et al. (2013), quienes demostraron que la suplementación 
dietética con curcumina aumentó la altura de las vellosidades del intestino delgado. Los autores 
sostienen que las vellosidades más altas proporcionan una mayor área de absorción de nutrientes, 
mientras que las vellosidades intestinales cortas o dañadas perjudican la capacidad de absorción 
debido a la disminución del área. 
En cuanto a la permeabilidad intestinal, el nivel sérico de isotiocianato de fluoresceína dextrán (FITC-
d) aumentó 3.26, 4 y 4.02 veces más en el grupo AF, en comparación con el grupo Control. Estos 
hallazgos concuerdan con lo reportado por Hernández-Ramírez et al. (2020), quienes reportaron un 
aumento significativo en la concentración sérica del FITC-d en pollos alimentados con una dieta 
contaminada con AFB1 (470 ng/g). Finalmente, la respuesta de hipersensibilidad cutánea a los 
basófilos (CBH) se incrementó en los grupos AF+alfalfa y AF+PCL, en comparación con el resto de los 
grupos experimentales.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Figura 5. Efecto del adsorbente de alfalfa y la AFB1 sobre la microbiota intestinal, la permeabilidad intestinal, 
y la altura de las vellosidades intestinales de los pavos. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
67 
 
Conclusiones  
 
La caracterización del adsorbente de las hojas de alfalfa permitió identificar el tipo de interacciones 
entre éste y la molécula de AFB1. Primeramente, los análisis FTIR-ATR, MEB, y EFRX revelaron, la 
presencia de diversos grupos funcionales, la morfología y la estructura superficial del adsorbente, y 
su composición elemental. En conjunto, los resultados de la caracterización, indican un papel crucial 
en el proceso de adsorción de la molécula de AFB1, especialmente en la capacidad de formar enlaces 
de hidrógeno con los átomos de oxígeno de la AFB1 en las tres secciones intestinales de las aves. En 
cuanto a las propiedades de carga del adsorbente, se concluyó que éste formó interacciones 
electrostáticas con la molécula de AFB1 principalmente bajo condiciones de pH de 2 y 5. Finalmente, 
el uso de la espectroscopía UV-Vis y la espectroscopía de fluorescencia permitieron cuantificar las 
clorofilas y los carotenoides presentes en el adsorbente, los cuales son cruciales en la adsorción 
debido a la formación de complejos clorofila- AFB1. 
En cuanto a los estudios de adsorción in vitro, el modelo pH-dependiente mostró una alta eficiencia 
de adsorción de AFB1, con valores superiores al 98.2% en los tres valores de pH evaluados (2, 5 y 7). 
Sin embargo, el modelo que emuló el TGI de las aves mostró una disminución en el porcentaje de 
adsorción, alcanzando un 88.8%. Finalmente, en el modelo in vivo, la suplementación de la dieta de 
los pavitos con el adsorbente de hojas de alfalfa mejoró significativamente los parámetros 
productivos, mientras que los valores de la bioquímica sanguínea no revelaron diferencias 
significativas en comparación con el grupo control. Respecto a la histología hepática, los dos grupos 
experimentales suplementados con las hojas de alfalfa no presentaron diferencias significativas en 
lesiones hepáticas en comparación con el grupo de control.  
En cuanto a la microbiota, la suplementación con el adsorbente de alfalfa promovió el crecimiento 
de bacterias benéficas como Faecalibacterium y Coprococcus catus, aumentó la altura de las 
vellosidades (771.3 μm), y mejoró significativamente la permeabilidad intestinal (214.8 ng/mL), en 
comparación con el grupo AF (858.2 ng/mL). Finalmente, el grupo AF+alfalfa tuvo un aumento en la 
respuesta de hipersensibilidad de los basófilos cutáneos (0.83 mm) en comparación con los demás 
grupos experimentales.  
El uso de un nivel bajo de inclusión del adsorbente derivado de las hojas de alfalfa (0.5% p/p) resultó 
altamente efectivo en la adsorción de AFB1 en ambos modelos in vitro. Esto se debió principalmente 
a la abundancia de clorofilas en el adsorbente, lo cual permitió la formación de complejos clorofila-
68 
 
AFB1. Además, las interacciones electrostáticas y no electrostáticas, otorgaron al adsorbente 
derivado de las hojas de alfalfa una notable eficacia en la adsorción de AFB1 presente en el alimento 
contaminado destinado para las aves. En términos generales, la incorporación del material 
adsorbente contrarrestó los efectos negativos de la AFB1 en las aves, principalmente por las diversas 
interacciones estudiadas en los modelos in vitro, sumadas a su posible efecto antioxidante, 
antiinflamatorio e inmunoestimulante en los pavos, favoreciendo la colonización del intestino por 
bacterias benéficas y mejorando la permeabilidad intestinal. Estos resultados, permiten concluir que 
el adsorbente de hojas de alfalfa es un material eficaz para remover AFB1 y, por tanto, mitigar sus 
efectos tóxicos en los pavos. 
Perspectivas 
Se considera necesario la realización de isotermas de adsorción con el adsorbente de alfalfa, ya que 
nos permiten conocer con mayor precisión la tasa de adsorción de la AFB1 en un período de tiempo 
y a una temperatura determinados. Además, sería importante llevar a cabo estudios de adsorción in 
vitro con el adsorbente de alfalfa para otras micotoxinas, las cuales son relevantes para la salud de 
animales y humanos. 
Finalmente, es imprescindible continuar investigando diversos productos orgánicos que se 
caractericen por tener una estructura porosa, contener mayores cantidades de clorofilas, ser una 
fuente potencialmente valiosa de antioxidantes y poseer una amplia variedad de grupos funcionales. 
 
 
 
 
 
 
 
 
 
69 
 
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