Centro de Investigación en Alimentación y
Desarrollo, A.C.
“ ACTIVIDAD ANTIHIPERTENSIVA DE LECHE FERMENTADA
CON CEPAS ESPECÍFICAS DE Lactococcus lactis “
POR:
JOSÉ CARLOS RODRÍGUEZ FIGUEROA
TESIS APROBADA POR LA
COORDINACIÓN DE TECNOLOGÍA DE ALIMENTOS
DE ORIGEN ANIMAL
COMO REQUISITO PARA OBTENER EL GRADO DE
DOCTORADO EN CIENCIAS
HERMOSILLO, SONORA DICIEMBRE DEL 2011
ii
APROBACIÓN
Los miembros del Comité designado para revisar la tesis de José Carlos
Rodríguez Figueroa, la han encontrado satisfactoria y recomiendan sea
aceptada como requisito parcial para obtener el grado de Doctor en Ciencias.
iii
iv
DECLARACIÓN INSTITUCIONAL
Se permiten y agradecen las citas breves del material contenido en ésta
tesis sin el permiso especial del autor, siempre y cuando se dé el crédito
correspondiente. Para la reproducción parcial o total de la tesis con fines
académicos, se deberá de contar con la autorización escrita del Director
General del Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD).
La publicación en comunicaciones científicas o de divulgación popular de
los datos contenidos en ésta tesis, deberá dar los créditos al CIAD, previa
aprobación por escrito del Director de la tesis.
v
DEDICATORA
A mis padre-madre divinos por darme la oportunidad de crecer en estado de consciencia y aportar a la humanidad
A Blanquita Angélica y Loskar Hyadi por habernos escogido como sus padres y por ser fuente de inspiración
A mi esposa Maura Marcela Ibarra Soto por su amor y porque juntos alcanzamos esta meta
A mi Mamá Blanca Esthela Figueroa Álvarez y a mi Papá José Carlos Rodríguez Laura por TODO su infinito amor
A mis hermanos Edgar Antonio y Ramsés por estar siempre presentes, lo mismo que mis abuelas, tías, tíos, primas y primos
A mis suegros Virginia Soto Federico y José Roberto Ibarra Borbón y familiares por todo su infinito cariño y apoyo
A toda nuestra familia humana
vi
AGRADECIMIENTOS
Al Consejo Nacional de Ciencia y Tecnología (CONACYT), por el apoyo a
través de los proyectos de investigación 42340-Z y 134295, además de la beca
otorgada para llevar a cabo mis estudios de doctorado.
Al Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD) por darme
la oportunidad de satisfacer mis inquietudes intelectuales y aportar un granito
de arena al umbral de la ciencia siempre pensando en la sociedad.
Enorme agradecimiento por apoyo, amistad y motivación a la Dra. Belinda
Vallejo Galland, mil gracias.
Muchas gracias al grupo mágico de Asesores Dra. Irasema Vargas Arispuro, Dr.
Hugo Sergio García Galindo y Dr. Humberto Astiazarán García que siempre
estuvieron aportando a nuestro trabajo de tesis, mil gracias por haberme guiado
en el sendero de la ciencia. Al Dr. Aarón F. González Córdova por haberme
invitado a la maestría inicialmente y apoyo durante mi estancia en CIAD.
Agradezco también a la Dra. María de Jesús Torres Llanez, al Dr. Miguel Angel
Mazorra Manzano. Al grupo de trabajo del Laboratorio de Lácteos, los M. en C.
Carmen Estrada Montoya y Ricardo Reyes, por el apoyo técnico en el uso del
equipo, así como a todos mis compañeros, MIL GRACIAS.
Al grupo de Marinos integrado por las M. en C. María Elena Lugo, Guillermina
García y Gisela Carvallo por compartir sus instalaciones. Lo mismo al equipo de
trabajo del Dr. Astiazarán, Q.B. Bertha I. Pacheco Moreno y M. en C. Ana
Cristina Gallegos, por su apoyo en la valoración de la actividad hipolipidémica,
gracias.
vii
Mil gracias a mis maestros: las doctoras Teresa Gollas, Juanita M. Meléndez,
Ana María Calderón, Luz Vázquez, Adriana Muhlia y los doctores Humberto
González, Rogerio Sotelo, Jesús Hernández, Martín Esqueda y Gustavo
González.
Muchas gracias a todos los que desde el anonimato de una u otra forma
hicieron que esta tesis fuera posible.
Un agradecimiento especial al encargado de la biblioteca Gerardo Reyna Canez
por facilitarme artículos de investigación, a la Ing. Karla Gabriela Robles Bernal
por su apoyo técnico. Asimismo agradezco a los Ingenieros Luis Alfonso Leyva
y Martín Peralta Contreras por sus aportaciones en la instalación y operación
del equipo para medir la presión arterial a las ratas. También agradezco a la Lic.
Laura Elizabeth García Cruz por su apoyo en la realización de los trámites
necesarios para la gestión de la beca doctoral CONACYT, a la Lic. Verónica
Araiza Sánchez por su colaboración en la realización de los trámites
relacionados con el pago de la beca, a Argelia Marín Pacheco por su apoyo en
la asignación de aulas para llevar a cabo las reuniones con el Comité de tesis, a
la Ing. Aurora Vidal Martínez por su apoyo técnico en la realización de
reuniones virtuales.
A todos mis amigos en especial a Vianey Trejo, Gaby Arreola, Ixchel Miranda,
Rebeca Jiménez, Laura y Adrian, Angel Valdés, y a nuestros compadres Pili y
Fernando, Maltie y Jaime, Gaby y Manuel por su apoyo incondicional. También
a todos mis compañeros del doctorado, en especial a mi generación Irlanda
Lagarda, Oliviert Martínez, Juan Pablo Valenzuela, Andre-i Sarabia por hacer
placenteros los momentos difíciles.
Y gracias también a mis amigos del club de ciclismo de montaña “Los
Cascabeles”.
viii
MUCHAS GRACIAS a las chicas de la clase de yoga Cuquis, Anita, Carmelita,
Mayra, Soco, Irene y Susy que siempre estuvieron en la mejor disposición de
apoyarme. También agradezco a todos los amigos GFU´cianos.
“No importa cuántos obstáculos haya que vencer, SIGUE ADELANTE y deja
que tu intuición y tu esencia fluyan hasta re-encontrarte con el sendero de la
Victoria…
“MÉXICO, CREO EN TÍ !!!
ix
ÍNDICE
Páginas
RESUMEN…………………….………………………..………...………....... ix
Capítulo 1. Integración general...……………………………….…………... 1
Capítulo 2. Angiotensin-converting enzyme inhibitory activity of milk
fermented by wild and industrial Lactococcus lactis strains…...………… 23
Capítulo 3. Novel angiotensin I-converting enzyme inhibitory peptides
in fermented milk by specific wild Lactococcus lactis strains.........……… 31
Capítulo 4. Specific wild Lactococcus lactis strains able to ferment milk
with relevant blood pressure and heart rate lowering effect...…………… 58
Capítulo 5. Antihypertensive and hypolipidemic effect of milk fermented
by specific Lactococcus lactis strains………............……………………… 79
x
RESUMEN
La Organización Mundial de la Salud señala que la hipertensión arterial se ha
convertido en un problema de salud mundial por su asociación con las
enfermedades cardiovasculares. En la actualidad, la alta presión arterial se
controla mediante el empleo de fármacos sintéticos, sin embargo, su uso está
vinculado a efectos secundarios. Ante las premisas anteriores aunadas al
carácter asintomático de la enfermedad, es necesario buscar alternativas
viables que permitan reducir la alta presión arterial de manera segura. Se ha
reportado que las proteínas de la leche son una fuente importante de
componentes bioactivos. Por otro lado, se ha demostrado que gracias al
complejo sistema proteolítico de las bacterias ácido lácticas, tal como
Lactococcus (L.) lactis, es posible producir péptidos antihipertensivos a partir de
las proteínas de la leche. Por lo anterior, el objetivo del presente trabajo
consistió en evaluar la actividad antihipertensiva de leches fermentadas con
cepas específicas de L. lactis. Para lo cual, se utilizaron veinte cepas de L.
lactis provenientes de diferentes nichos ecológicos. Posteriormente, se
fermentó leche para seleccionar las cepas con la mayor capacidad proteolítica y
actividad antihipertensiva. Se obtuvieron los extractos acuosos de las leches
fermentadas de las cepas específicas de L. lactis y se fraccionaron mediante
cromatografía líquida de alta resolución (HPLC). Por un lado, se identificaron 37
nuevas secuencias peptídicas a través de HPLC acoplada a espectrometría de
masas, mientras que por el otro, se evaluó la actividad antihipertensiva in vitro
de las diferentes fracciones peptídicas. Además, se procedió a evaluar la
actividad antihipertensiva en un modelo murino. Para esto, se seleccionaron las
cepas que presentaron las fracciones peptídicas con la mayor capacidad
antihipertensiva L. lactis NRRL B-50571 y B-50572, para evaluar la disminución
xi
de la presión arterial y pulso cardiaco en ratas espontáneamente hipertensas
(SHR). Los extractos acuosos de las leches fermentadas fueron dosificados vía
oral en dos concentraciones de proteína, 35 y 50 mg de proteína por kilogramo
de peso del animal. Todos los extractos acuosos de las leches fermentadas
fueron capaces de disminuir la presión arterial así como el pulso cardiaco. La
máxima reducción de la presión arterial sistólica (17.7 ± 4 y mm Hg) se observó
en las SHR que recibieron el extracto acuoso, con 35 mg de proteína por
kilogramo de peso del animal, de las leches fermentadas con L. lactis NRRL B-
50571. Por otro lado, el extracto acuoso (50 mg proteína por kilogramo de peso
del animal) obtenido a partir de la leche fermentada con L. lactis NRRL B-50572
mostró la máxima disminución de la presión arterial sistólica (23.9 ± 9.4 mm
Hg). En el caso del pulso cardiaco, los extractos acuosos de las leches
fermentadas con L. lactis NRRL B-50572 y B-50571 correspondientes a las
concentraciones antes mencionadas, fueron capaces de disminuir 16.9 ± 11.5 y
16.6 ± 9.2 pulsos min-1, respectivamente. Adicionalmente, la ingesta de las
leches fermentadas con L. lactis NRRL B-50571 y L. lactis NRRL B-50572
mejoraron el perfil lipídico de las SHR, ya que fueron capaces de reducir el
colesterol de baja densidad en plasma. Estos resultados muestran la capacidad
que tienen las cepas específicas de L. lactis para producir leche fermentada con
capacidad para disminuir la alta presión arterial y el pulso cardiaco in vivo, al
igual que la concentración de colesterol de baja densidad. En conclusión, las
leches fermentadas con cepas específicas L. lactis constituyen un alimento
lácteo funcional con potencial para ser utilizado en la prevención y/o
coadyuvante para mejorar la salud cardiovascular.
Capítulo 1
Integración general
2
INTRODUCCIÓN
Los malos hábitos alimentarios y el estilo de vida aunado a factores
genéticos han hecho de la hipertensión un problema de salud mundial (Mataix,
2002, Chobanian, 2004).
La hipertensión consiste en el aumento de la presión arterial, lo que
dificulta la disponibilidad de nutrientes y de oxígeno a la célula. Esta
padecimiento se asocia a enfermedades cardiovasculares, diabetes, infarto al
miocardio, enfermedades vasculares cerebrales trombóticas y hemorrágicas. La
ausencia de síntomas en los pacientes con hipertensión, convirtió a esta
enfermedad en asintomática, por lo cual se le considera “el asesino silencioso”.
Este padecimiento no es curable, pero sí controlable (Mataix, 2002; Chobanian,
2004).
Los tratamientos contra la hipertensión enfatizan su atención contra los
factores que la detonan, sin embargo también se recurre al uso de fármacos.
Los más modernos son los bloqueadores adrenérgicos e inhibidores de la
enzima convertidora de la angiotensina (ECA) (McPhee et al., 2001). Estos
últimos interrumpen la transformación de angiotensina I en angiotensina II y con
ello evitan la vasoconstricción arterial (Guan-Hong et al., 2004).
Asimismo, existen alimentos que además de aportar nutrientes, ofrecen
péptidos con diversas actividades biológicas, denominados funcionales (Diplock
et al., 1999). Korhonen y Pihlanto (2006), señalaron en su revisión bibliográfica
la presencia de péptidos bioactivos en alimentos lácteos fermentados y
demostraron que estos benefician a los sistemas cardiovascular, digestivo,
inmunológico y nervioso.
3
La actividad antihipertensiva de algunos de estos péptidos ha sido
demostrada tanto in vivo como in vitro (Gómez-Ruiz et al., 2004a; Muguerza et
al., 2006).
Aún cuando los péptidos antihipertensivos pueden ser producidos por
hidrólisis proteica con enzimas digestivas y por el procesamiento del alimento,
es la fermentación con cultivos proteolíticos iniciadores, principalmente las
bacterias ácido lácticas (BAL), la opción más simple y segura de generarlos
(Korhonen y Pihlanto, 2003; Korhonen y Pihlanto, 2006; Donkor et al., 2007).
Las BAL poseen un complejo metabolismo que condiciona su actividad
proteolítica, la generación de ácido láctico y la producción de bacteriocinas. Es
por ello que las características organolépticas, tecnológicas y nutricionales del
alimento dependen de la presencia de las cepas. La contribución en dos o más
características antes mencionadas permite clasificar a las BAL como
multifuncionales (Leroy y De Vuyst, 2004). Lactococcus (L.) lactis es un ejemplo
de BAL multifuncional por generar péptidos bioactivos compuestos
responsables de aroma y por determinar la textura de productos lácteos
fermentados (Pihlanto-Leppälä et al., 1998; Leroy y De Vuyst, 2004).
L. lactis es utilizado ampliamente como cultivo iniciador comercial, por lo
que económicamente es importante (Savijoki et al., 2006). Además se
encuentra extensamente distribuido en la naturaleza (Topisirovic et al., 2006).
De acuerdo con Ayad et al. (1999), la generación de compuestos volátiles está
influenciada por el origen del aislamiento de las cepas de L. lactis. Sin embargo,
su capacidad para producir péptidos potencialmente antihipertensivos y la
repercusión que podría tener el origen del aislamiento de L. lactis ha sido poco
investigada.
4
Estudios recientes señalaron que cepas de L. lactis aisladas de su nicho
ecológico, particularmente de leche cruda fermentada, pueden generar péptidos
con actividad inhibidora de la enzima convertidora de angiotensina, capaces de
disminuir la hipertensión arterial, en leche fermentada (Muguerza et al., 2006).
Asimismo, se ha encontrado que estos microorganismos pueden generar
péptidos potencialmente antihipertensivos en queso fresco (Torres-Llanez,
2007). Esto permite considerar la presencia de péptidos inhibidores de la
enzima convertidora de la angiotensina en leche fermentada con cepas de L.
lactis aislados de diversos orígenes. Sin embargo, es necesario evaluar la
capacidad que presenta la leche fermentada para disminuir la presión arterial y
el pulso cardiaco in vivo.
Los estudios enfocados a valorar el efecto hipotensivo de alguna
substancia de interés, tal como los péptidos derivados de proteínas lácteas,
recurren al uso del modelo murino. En los últimos 10 años, la base de datos
MEDLINE contabilizó un total de 5, 059 trabajos realizados con ratas
espontáneamente hipertensas (SHR). Estos datos muestran a dicha cepa como
la cepa de mayor interés para estudiar la actividad antihipertensiva. Hasta el
momento, las SHR fungen como el modelo animal que mejor similitud
ejemplifica a la hipertensión arterial esencial en humanos (Pravenec y Kurtz
2010).
Por lo anterior, el objetivo de esta investigación se centra en evaluar la
actividad antihipertensiva de leches fermentadas con cepas específicas de L.
lactis tanto in vitro como in vivo, así como identificar las secuencias peptídicas
asociadas a dicha bioactividad.
5
JUSTIFICACIÓN
Ante el incremento de personas con hipertensión, el carácter asintomático de la
enfermedad y los efectos secundarios que conlleva el uso de fármacos, es
necesario explorar nuevas opciones que sean capaces de disminuir la alta
presión arterial de manera segura. Por lo que la ingesta de péptidos
antihipertensivos a partir de leche fermentada con cepas específicas de L.
lactis, podría favorecer el manejo de la hipertensión arterial.
HIPÓTESIS
La leche fermentada con cepas específicas de L. lactis presenta actividad
antihipertensiva en ratas espontáneamente hipertensas (SHR).
6
OBJETIVOS
Objetivo General
Evaluar la actividad antihipertensiva de leches fermentadas con cepas
específicas de L. lactis.
Objetivos Específicos 1.- Aislar las fracciones peptídicas de los extractos acuosos ˂ 3 kDa.
2.-Identificar las secuencias de los péptidos asociados a la actividad
antihipertensiva.
3.- Evaluar la actividad hipotensiva de la leche fermentada con cepas
específicas de L. lactis en un modelo murino.
7
METODOLOGÍA
Etapa I. Evaluación de la actividad inhibitoria de la enzima convertidora de la
angiotensina (IECA) de leche fermentada con cepas específicas de L. lactis.
Etapa II. Valoración del efecto antihipertensivo de la leche fermentada con cepas específicas de L. lactis en un modelo murino mediante una sola dosis.
Fermentación de la
leche con L. lactis
Obtención de los extractos
acuosos (EA) ˂ 3 kDa
Fraccionamiento de
los EA por HPLC-FR1
Evaluación de la IECA Identificación de las
fracciones peptídicas
asociados a la
actividad IECA
Fermentación de la leche con L. lactis
Obtención de los extractos acuosos
Intubación intragástrica de los
animales
Medición del efecto antihipertensivo
8
1 Cromatografía Líquida de Alta Resolución en Fase Reversa
Etapa III. Estudio del efecto antihipertensivo de leche fermentada con cepas
específicas de L. lactis en ratas espontáneamente hipertensas (SHR) a largo
plazo.
Fermentación de la
leche con L. lactis
Obtención de las
muestras
Libre acceso de las SHR
a los tratamientos
Medición del efecto
antihipertensivo y
evaluación del perfil
lipídico
9
DESCRIPCIÓN DE LOS CAPÍTULOS
El Capítulo 1 integra todo el trabajo desarrollado de manera general. Este
capítulo aporta la estructura de la tesis considerando la introducción a la
temática, justificación del estudio, así como el planteamiento de la hipótesis
aunado a los objetivos general y particulares. Asimismo, también se incluyen las
conclusiones y perspectivas del trabajo. Finalmente, se anexan las referencias
consultadas durante el mismo.
En el Capítulo 2 se describe la actividad inhibitoria de la enzima
convertidora de la angiotensina (IECA) de las leches fermentadas con cepas
específicas de L. lactis. En este apartado se incluye el estudio en donde se
utilizaron veinte cepas pertenecientes a diferentes nichos ecológicos. Asimismo,
se reporta la mayor IECA en los extractos acuosos ˂ 3 kDa obtenidos de las
leches fermentadas con las cepas aisladas de lácteos artesanales y cultivos
comerciales.
El Capítulo 3, se reportan nuevas secuencias peptídicas asociadas a la
actividad IECA. Tales secuencias fueron encontradas en los extractos acuosos
de la leche fermentada con cepas específicas de L. lactis. Por otro lado, en el
Capítulo 4 se presentan los efectos reductores de la alta presión arterial y el
pulso cardiaco de los extractos acuosos en ratas espontáneamente hipertensas
(SHR) a corto plazo.
Finalmente, el Capítulo 5 muestra la evaluación de la actividad
antihipertensiva de la leche fermentada con cepas específicas de L. lactis. En
este estudio se reporta la disminución de la presión arterial en las SHR durante
las cuatro semanas de experimentación.
10
CONCLUSIÓN Y PERSPECTIVAS
En conclusión, las leches fermentadas con cepas específicas de L. lactis
presentaron una importante capacidad para inhibir la actividad de la enzima
convertidora de la angiotensina. Asimismo, los estudios in vivo, tanto a corto
como a largo plazo, demostraron que la ingesta de las leches fermentadas con
cepas específicas de L. lactis tiene relevantes efectos reductores de la presión
arterial, del pulso cardiaco y del colesterol de baja densidad. Además, se
identificaron 37 nuevos péptidos asociados a la actividad antihipertensiva.
Es recomendable considerar futuros estudios in vivo con una sola dosis
en donde se evalúe la capacidad hipotensiva de los péptidos presentes en las
fracciones peptídicas con mayor actividad, de tal manera que se pueda dilucidar
la relación entre péptido antihipertensivo-efecto hipotensivo. En este mismo
tenor, una vez identificados los péptidos capaces de disminuir la presión arterial
en mayor magnitud, sería importante estudiar el mecanismo de absorción de los
mismos utilizando péptidos marcados.
Por otro lado, también se sugiere realizar estudios enfocados a conocer
los sistemas proteolítico y peptidolítico de las cepas L. lactis que permitan
asociar diferentes actividades enzimáticas a la generación de secuencias
peptídicas con potencial antihipertensivo.
11
REFERENCIAS
Aleksandrzak-Piekarczyk, T., Kok, J. Renault, P. y Bardowski, J. 2005.
Alternative lactose catabolic pathway in Lactococcus lactis IL1403.
Appl. Envirom.Microbiol. 71:6060-6069.
Algaron, F., Miranda, G., Le Bars, D. y Monnet, V. 2004. Milk fermentation by
Lactococcus lactis with modified proteolytic systems to accumulate
potentially bio-active peptides. Lait 84:115-123.
American Dietetic Association (ADA) (2004). “ Position of the American Dietetic
Association: Functional foods”. J. American Dietetic Association
104:814-826.
Ashar, M.N y Chand, R. 2004. Fermented milk containing ACE-inhibitory
peptides reduces blood pressure in middle aged hypertensive subjects.
Milchwissenchaft 59:363-366.
Ayad, E.H.E., Verheul, A., Jong, C., Wouters, J.T.M. y Smit, G. 1999. Flavour
forming abilities and amino acid requirements of Lactococcus lactis
strains isolated from artisanal and non-dairy origin. Int. Dairy J. 9:725-
735.
Ayad, E., Verheul, A., Engels, W., Wouter, J., y Smit, G. 2001. Enhanced flavour
formation by combination of selected lactococci from industrial and
artisanal orign with focus on completion of a metabolic pathway. J. Appl.
Microbiol. 90:59-67.
Ayad, H., Nashat, S., El-Sadek, Metwaly, H. y El-Soda. 2004. Selection of wild
lactic acid bacteria from traditional Egyptian dairy products according
to production and technological criteria. Food Microbiol. 21:715-725.
Ayad, E.H.E. Verheul, A., Wouters, T.M. y Smit, G. 2000. Application of wild
starter cultures for flavour development in pilot plant cheese making. Int.
Dairy J. 10:169-179.
12
Baranyi, J. y Roberts, T. 1994. A dynamic approach to predicting bacterial
growth in food. Int. J. Food Microbiol. 23:277-294.
Barber Fox, M. y Barber Gutiérrez, E. 2003. El Sistema renina-angiotensina y el
riñón en la fisiopatología de la hipertensión arterial esencia. Rev. Cubana
Invest Biomed. 22(3): 192-198.
Beresford, T., Fitzsimons, N., Brennan, N. y Cogan, T. 2001. Recent advances
in cheese microbiology. Int. Dairy J. 11:259-274.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein dye
binding. Anal. Biochem. 72:248-254.
Burrows, R., Nettleton, S. y Bunton, R. 1995. Sociology & health promotion.
Health, risk and consumption under late modernism. In The Sociology of
Health Promotion. (ed. by R. Bunton, S. Nettleton & R. Burrows). Pp 1-9.
Routledge, London. Citado en: Niva, M. y Mäkelä, J. 2007. Finns and
functional foods:socio-demographics, health efforts, notions of technology
and the acceptability of health-promoting foods. Int. J. Consumer Studies
31:34-45.
Centeno, J., Gaya, P., Medina, M., y Núñez, M. 2002. Cross-inhibition among
wild strains of Lactococcus lactis isolated from the same ecological niche
J. Food Protection 65:205-210.
Cheung, H.S., Feng-Lai, W., Ondetti, M.A., Sabo, E.F. y Cushman, D.W. 1980.
Binding of peptide substrates and inhibitors of angiotensin-converting-
enzyme. J. Biol. Chem. 255:401-407.
Chobanian, A.V. 2004. The seventh report of the joint national committee on
prevention, detection, evaluation and treatment of high blood pressure.
US Department of Health and Human Services. Pp. 25-32.
13
Christensen, J.E., Dudley, E.G., Pederson, J.A. y Steele, J.L. 1999. Peptidases
and amino acid catabolism in lactic acid bacteria. Antonie van
Leeuweenhoek. 76:217-246.
Church, F., Swaisgood, H.E., Porter, D., Catignani, G.L. 1983.
Spectrophotometric assay using o-phthaldialdehyde for determination of
proteolysis in milk and isolated milk proteins. J. Dairy Sci. 66:1219-1227.
Consejo Europeo de Información sobre Alimentación (EUFIC). 2007.
http://www.drluengo.net/Alimentos%20Funcionales.htm Consultada:
7/11/07.
Cushman, D. y Cheung, H. 1971. Spectrometric assay and properties of the
angiotensin converting enzyme of rabbit lung. Biochemical Pharmacology
20:1637-1648.
Deegan, L., Cotter, P., Hill, C. y Ross, P. 2006. Bacteriocins: Biological tools for
bio-preservation and shelf-life extension. Int. Dairy J. 16:1058-1071.
Dent, M.P., O´Hagan, S., Braun, W.H., Schaetti, P., Marburger, A. y Vogel, O.
2007. A 90-day subchronic toxicity study and reproductive toxicity Studies
on ACE-inhibiting lactotripeptide. Food Chem. Toxicology 45:1468-1477.
Diplock, A.T., Agged, P.J., Ashwell, M., Bornet, F., Fern, E.B. & Roberfroid, M.B.
1999. Scientific concepts of functional foods in Europe: consensus docu-
ment. British J. Nutr. 81: S1-S27.
Doman-Pytka, M. Renault, P. y Bardowski, J. 2004. Gene-cassette for
adaptation of Lactococcus lactis to a plant environment. Lait. 84:33-37
Donkor, O.N., Henriksson, A., Sigh, T.K., Vasiljevic, T. y Shah, N.P. 2007. Ace-
inhibitory activity of probiotic yoghurt. Int. Dairy J. 17:1321-1331.
FitzGerald, R.J. y Murray, B.A. 2006. Bioactive peptides and lactic
fermentations. Int. J. Dairy Technol. 59:118-125.
FitzGerald, R. J., Murray, B. y Walsh, D. 2004. Hypotensive peptides from milk
proteins. J. Nutr. 134: 980S-988S.
14
FitzGerald, R. J. y Meisel, H. 2000. Milk protein-derived peptide inhibitors of
angiotensin-I-converting enzyme. British J. Nutr. 84:1:S33-S37.
Fuglsang, A., Rattray, F.P., Nilsson, D. y Nyborg, N.C.B. 2003. Lactic acid
bacteria: inhibition of angiotensin converting enzyme in vitro and in vivo.
Antonie van Leeuwenhoek. 83:27-34.
García, M. 1986. Leches fermentadas como vehículos de probióticos. Archivos
de Investigación Pediátrica de México. Suplemento Especial: Los
Probióticos en la nutrición
http://www.medinet.net.mx/conapeme/revistas/Suplemento%20Nut
ricion/ Leches_fermentadas.htm Consultada: 8/11/07.
Gaudix, A., Gaudix, E., Páez-Dueñas, M.P., González-Tello, P. y Camacho, F.
2000. Procesos tecnológicos y métodos de control en la hidrólisis de
proteínas. Ars. Pharmaceutical 41:1; 79-89.
Gobbetti, M., Ferranti, P., Smacchi, E., Goffredi, F. y Addeo, F. 2000. Production
of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks
started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and
Lactococcus lactis subsp. cremoris FT4. Appl. Environ. Microbiol.
66:3898-3904.
Gómez-Ruiz, J.A., Ramos, M. y Recio, I. 2004a. Angiotensin converting
enzyme- inhibitory activity of peptides isolated from Manchego
cheese. Stability under simulated gastrointestinal digestion. Int. Dairy
J. 14:1075-1080.
Gómez-Ruiz, J.A., Ramos, M. y Recio, I. 2004b. Identification and formation of
angiotensin-converting enzyme.inhibitory peptides in Manchego cheese
by high-performance liquid chromatography-tandem mass spectrometry.
J. Chromatography 1054:269-277.
15
Guan-Hong, L. Guo-Wei, L. Yong-Hui, S. y Sundar, S. 2004. Angiotensin I-
converting enzyme inhibitor peptides derived from food proteins and their
physiological and pharmacological effects. Nutr. Research. 24:469-486.
Gutiérrez-Méndez, N., Vallejo-Córdoba, B., González-Córdova, A., Nevárez-
Moorillón, G.V. y Rivera-Chavira, B. 2008. Evaluation of aroma
generation of Lactococcus lactis with an electronic nose and sensory
analysis. J. Dairy Sci. J. Dairy Sci. 91:49-57.
Hata, Y., Yamamoto, M., Ohni, M., Nakajima, K., Nakamura, Y. y Takano, T.
1996. A placebo-controlled study of the effect of sour milk on blood pres-
sure in hypertensive subjects. Am. J. Clin. Nutr. 64:767-771.
Hartmann, R. y Meisel, H. 2007. Food-derived peptides with biological activity
from research to food applications. Curr. Opinion in Biotechnol.
18:163-169.
Hernández-Ledesma, B., Amigo, L., Ramos, M. y Recio, I. 2004. Angiotensin
converting enzyme inhibitory activity in comercial fermented products for-
mation of peptides under simulated gastrointestinal digestion. J. Agric.
Food Chem. 52:1504-1510.
Hipertensión arterial, 2008.
http://www.dmedicina.com/salud/corazon/hipertension-arterial.html
Consultada: 25/3/08.
Inoue, K., Shirai, T., Ochiai, H., Kasao, M., Hayakawa, K., Kimura, M. y
Sansawa, H. 2003. Blood-pressure-lowering effect of a novel fermented
milk containing ɣ -aminobutyric acid (GABA) in mild hypertensives. Eur. J.
Clin. Nutr. 57:490-495.
Jauhiainen, T., Vapaatalo, H., Poussa, T., Kyrönpalo, S., Rasmussen, M. y
Korpela, R. 2005. Lactobacillus helveticus fermented milk lowers blood
pressure in hypertensive subjects in 24-h ambulatory blood pressure
measurement. American J. Hipertension. 18:1600-1605.
16
Kawase, M., Hashimoto, H., Hosoda, M., Morita, H. y Hosono, A. 2000. Effect of
Administration of fermented milk containing whey protein concentrate to
rats and healthy men on serum lipids and blood pressure. J. Dairy Sci.
83:255-253.
Kern, M. 2002. Food, feed, fiber, fuel and industrial products of the future: chal-
lenges and opportunities. Understanding the strategic potential of plant
genetic engineering. J. Agronomy & Crop Sci. 188:291-305.
Khatib, O., MN. y El-Guindy, M.S. 2005. Clinical guidelines for the management
of hypertension. EMRO Technical Publications Series 29. WHO. Pp. 5-8.
Kitts, D., y Weiler, K. 2003. Bioactive proteins and peptides from food sources.
Applications of bioprocesses used in isolation and recovery. Curr.
Pharmaceutical Design 9:1309-1323.
Klijn, N., Weerkamp, A., y De Vos W. 1995. Detection and characterization of
lactose-utilizing Lactococcus spp. in natural ecosystems. Appl. Environ.
Microbiol. 61(2):788-792.
Konings, W.N. 2002. The cell membrane and the struggle for life of lactic acid
bacteria. Antonie van Leeuwenhoek. 82:3-27.
Korhonen, H. y Pihlanto, A. 2003. Food-derived bioactive peptides- opportunities
for designing future foods. Curr. Pharmaceutical Design 9:1297-1308.
Korhonen, H. y Pihlanto, A. 2006. Bioactive peptides: Production and
functionality. Int. Dairy J. 16:945-960.
Lee, Y. 1992. Food-processing approaches to altering allergenic potential of
milk-based formula. J. Pediatr. 121:S47-S50.
Leroy, F. y De Vuyst, L. 2004. Lactic acid bacteria as functional starter cultures
for the food fermentation industry. Trends in Food Sci & Technol. 15:67
-78.
17
Li, G., Le, G., Shi, Y. y Shrestha, S. 2004. Angiotensin I-converting enzyme inhi-
bitory peptides derived from food proteins and their physiological and
pharmacological effects. Nutr. Research 24:469-486.
Li, R., Nix, D. y Schentag, J. 1993. New turbidimetric assay for quantitation of
viable bacterial densities. Antimicrobial Agents and Chemotherapy
37:2:371-374.
López-Fandiño, R., Otte, J. y Van Camp. J. 2006. Physiology, chemical and tech
technological aspects of milk - protein-derived peptides with antihyperten-
sive and ACE-inhibitory activity. Int. Dairy J. 16:1277-1293.
Manrique, G. 2002. Alimentos funcionales. Una nueva era en la historia de la
alimentación.
www.mdp.edu.ar/rectorado/secretarias/investigacion/nexos/19/19-4.htm
Última actualización: 23/12/2002. Consultada: 7/11/2007.
Mataix, J. 2002. Nutrición y Alimentación Humana. Vol. II. Ed. Océano.
Barcelona, España. Pp.1145-1150.
Matsui, T., Tamaya, K., Seki, E., Osajima, K., Matsumoto, K., y Kawasaki, T.
2002. Val-Tyr as natural antihypertensive dipeptide can be absorved into
the human circulatory blood system. Clin. Exp. Pharmacol. Physiol.
29:204-208.
McPhee, S., Lingappa, V., Ganong, W. y Lange, J. 2001. Fisiopatología médica:
Una introducción a la medicina clínica. 3ra edición. Manual moderno.
México. Pp. 312-322 y 583.
Meisel, H., Goepfert, A. y Gunther, S. 1997. ACE inhibitory activities in milk
products. Milchwissenschaft 52:307-311.
Meisel, H. y FitzGerald, R.J. 2003 Biofunctional peptides from milk proteins:
Mineral binding and cytomodulatory effects. Curr. Pharmaceutical Design
9: 1289-1295.
18
Mitra, S., Chakrabartty, K. y Ranjan, S. 2005. Production and Characterization
of nisin-like peptide produced by a strain of Lactococcus lactis isolated
from fermented milk. Current Microbiol. 51:183-187.
Mizuno, S., Matsuura, K., Gotou, T., Nishimura, S., Kajimoto, O., Yabune, M.,
Kajimoto, Y. y Yamamoto, N. 2005. Antihypertensive effect of casein
hydrolysate in a placebo-controlled study in subjects with high-normal
blood pressure and mild hypertension. British J. Nutr. 94:84-91
Mizushima, S., Ohshige, K., Watanabe, J., Kimura, M., Kadowaki, T.,
Nakamura, Y., Tochlkubo, O. y Ueshima, H. 2004. Randomized
controlled trial of sour milk on blood pressure in borderline hypertensive
men. American J. Hypertension 17:701-706.
Morales, P., Fernández-García, E., Gaya, P., Medina, M. y Nuñez, M. 2001.
Hydrolysis of caseins and formation of hydrophilic and hydrophobic
peptides by wild Lactococcus lactis strains isolated from ewe´s milk
cheese. J. Appl. Microbiol. 91:907-915.
Motaghi, M., Mazaheri, M., Moazami, N., Farkhondeh, A., Fooladi, M.H. y
Goltapeh, E.M. 1997. Kefir production in Iran. World J. Microbiol. &
Biotechnol. 13:579-581.
Muguerza, B., Ramos, M., Sánchez, E., Manso, M., Miguel, M., Aleixandre, A.,
Delgado, M. y Recio, I. 2006. Antihypertensive activity of milk fermented
by Enterococcus faecalis strains isolated from raw milk. Int. Dairy J.
16:61-69.
Nakamura, Y., Yamamoto, N., Sakai, K. y Takano, T. 1995. Antihypertensive
effect of sour milk and peptides isolated from it that are inhibitors to
angiotensin I-converting enzyme. J. Dairy Sci. 78:1253-1257.
Niva, M. y Mäkelä, J. 2007. Finns and functional foods: socio-demographics,
health efforts, notions of technology and the acceptability of health
promoting foods. Int. J. Consumer Stud. 31:34-45.
19
Norma Oficial Mexicana NOM-185-SSA1-2002. Mantequilla, cremas, lecheras,
fermentados y acidificados. Especificaciones sanitarias. Diario Oficial de
la Federación. Pp. 5.
Ondetti, M., Williams, N., Sabo, E., Pluščec, Weaver, E. y Kocy, O. 1971. Angio-
tensin-converting enzyme inhibitors from the venom of Bothrops jaraca
isolation, elucidation of structure and synthesis. Biochemistry 10:4033
-4039.
Pravenec M., y Kurtz T. 2010. Recent advances in genetics of spontaneously
hypertensive rat. Curr. Hypertens. Rep., 12: 5-9.
Pihlanto-Leppälä, A., Rokka, T., Korhonen, H. 1998. Angiotensin I converting
enzyme inhibitory peptides derived from bovine milk proteins. Int. Dairy J.
8:325-331.
Quirós, A., Hernández-Ledesma, B., Ramos, M., Amigo, L. y Recio, I. 2005.
Angiotensin-converting enzyme inhibitory activity of peptides derived from
caprine kefir. J. Dairy Sci. 88:3480-3487.
Quirós, A., Ramos, M., Muguerza, B., Delgado, M., Miguel, M., Alixandre, A. y
Recio, I. 2007. Identification of novel antihypernensive peptides in milk
fermented with Enterococcus faecalis. Int. Dairy J. 17:33-41.
Raynaud, S., Perrin, R., Cocaing-Bousquet, M., y Loubiere, P. 2005. Metabolic
and transcriptomic adaptation of Lactococcus lactis subsp. lactis Biovar
diacetylactis in response to autoacidification and temperature downshift in
skin milk. Appl. Environ. Microbiol. 71(12): 8016-8023.
Salminen, S., Wright, A., y Ouwehand, A. 2004. Lactic Acid Bacteria: Microbiolo-
gical and functional aspects. 3er. Ed. New York. Pp. 1-5.
Sanders, J.W., Venema, G. y Kok, J. 1999. Environmental stress responses in
Lactococcus lactis. FEMS Microbiol. Rev. 23:483-501.
Savijoki, K., Ingmer, H. y Varmanen, P. 2006. Proteolytic systems of lactic acid
bacteria. Appl. Microbiol. Biotechnol. 71:394-406.
20
Seppo, L., Jauhiainen, T., Poussa, T. y Korpela, R. 2003. A fermented milk high
in bioactive peptides has a blood pressure-lowering effect in hypertensive
subjects. Am J. Clin. Nutr.77:326-330.
Seppo, L., Kerojoki, O., Suomalainen, T., y Korpela, R. 2002. The effect of a
Lactobacillus helveticus LBK-16H fermented milk on hypertension-a pilot
study on humans. Milchwissenschaft. 57:124-127.
Settanni, L. y Corsetti, A. 2008. Application of bacteriocins in vegetable Food
bio-preservation. Int. J. Food Microbiol. 121:123-138.
Singh, T., Drake, M., y Cadwaller, K. 2003. Flavor of Cheddar Cheese: A
chemical and sensory perspective. Comprehensive Reviews in Food Sci
and Food Safety. Vol. 2:166-189.
Smit, G., Smit, B.A. y Engels W.J.M. 2005. Flavour formation by lactic acid
bacteria and biochemical flavour profiling of cheese products. FEMS
Microbiol. Reviews 29:591-610
Soomro, A., Masud, T. y Anwaar, K. 2002. Role of lactic acid bacteria (LAB) in
food preservation and human health-A review. Pakistan J. Nutr. 1(1):20
-24.
Suetsuna, K. 1998. Isolation and characterization of angiotensin I-converting
enzyme inhibitor di-peptides derived from Allium sativum L.
(garlic).J.Nutr. Biochem. 9:415-419.
Todorov, S., Svetla, D., Van Reenen, C., Meincken, M., Dinkova, G., Ivanova, I.
y Dicks, L. 2006. Characterization of bacteriocin HV219, produced by
Lactococcus lactis subsp. lactis HV219 isolated from human vaginal
secretions. J. Basic Microbiol. 46:3:226-238.
Topisirovic, L., Kojic, M., Fira, D., Golic, N., Strahinic, I. y Lozo, J. 2006.
Potential of lactic acid bacteria from specific natural niches in food
production and preservation. Int. J. Food Microbiol. 112: 230-235.
21
Torres-Llanez, M.J. 2007. Aislamiento, caracterización e identificación e
péptidos potencialmente antihipertensivos en queso fresco inoculado con
bacterias ácido lácticas específicas. [Tesis de Doctorado en Ciencias]
Centro de Investigación en Alimentación y Desarrollo, A.C.; Hermosillo,
Sonora, México.
Toumilehto, J., Lindström, J., Hyyrynen, J., Korpela, R., Karhunen, M-L.,
Mikkola, L., Jauhiainen, T., Seppo, L. y Nissinen, A. 2004. Effect of
ingesting sour milk fermented using Lactobacillus helveticus bacteria
producing tripeptides on blood pressure in subjects with milk
hypertension. J. Human Hypertension. 18:795-802.
Van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S.D. y
Maguin, E. 2002. Stress responses in lactic acid bacteria. Antonie van
Leeuwenhoek. 82:187-216.
Verhagen, H., Coolen, S., Duchateau, G., Hamer, M., Kyle, J. y Rechner, A.
2004. Assessment of the efficacy of functional food ingredients
introducing the concept “kinetics of biomarkers”. Mutant Res. 551(1-2):65
-78.
Vermeirssen, V., Van Camp, J. y Verstraete. 2002. Optimization and validation
of an angiotensin-converting enzyme inhibition assay for the screening of
bioactive peptides. J. Biochim. and Biophysical Methods 51:75-87.
Vermeirssen, V., Van Camp, J. y Verstraete. 2004. Bioavailability of angiotensin
I converting enzyme inhibitory peptides. British J. Nutr. 92:357-366.
Yamamoto, N., Maeno, M. y Takano, T. 1999. Purification and characterization
of an antihypertensive peptide from a yoghurt-like product fermented by
Lactobacillus helveticus CPN4. J Dairy Sci 82:1388-1393.
Yamamoto, N., Ejiri, M. y Mizuno, S. 2003. Biogenic peptides and their potential
use. Current Pharmaceutical Design 9:1345-1355.
22
Walstra, P., Geurts, T., Noomen, A., Jellema, A. y Boekel, M. 1999. Dairy
Technology, Principles of milk properties and processes. Marcel, Dekker,
Inc. New York. Pp. 3-10
Wei, L., Alhenc-Gelas, F., Corvol, P. y Clauser, E. 1991. The two homologous
domains of human angiotensin-I-converting enzyme are both catalytically
active. J. Biol. Chem. 266:9002-9008.
Willen-Sanders, J., Venema, G. y Kok, J. 1999. Environmental stress responses
in Lactococcus lactis. FEMS Microbiol. Rev. 23:483-501.
Capítulo 2
Angiotensin-converting enzyme inhibitory
activity of milk fermented by wild and
industrial Lactococcus lactis strains
Artículo publicado en: Journal of Dairy Science, vol. 93, pp. 5032-5038
24
25
26
27
28
29
30
Capítulo 3
Novel angiotensin I-converting enzyme
inhibitory peptides in fermented milk by
specific wild Lactococcus lactis strains
Artículo enviado al: Journal of Dairy Science
32
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MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
33
Novel angiotensin I-converting enzyme inhibitory peptides produced in 1
fermented milk by specific wild Lactococcus lactis strains 2
3
4
J.C. Rodríguez-Figueroa, M.J. Torres-Llanez, A.F. González-Córdova, H. S. 5
Garcia, B. Vallejo-Cordoba* 6
7
8
Centro de Investigación en Alimentación y Desarrollo A.C. (CIAD). Carretera a la 9
Victoria Km. 0.6, Hermosillo, Sonora, 83000, México 10
* Corresponding Author. 11
Belinda Vallejo-Cordoba 12
Phone: +52 (662) 289-24-00 ext. 303; 13
Fax: +52 (662) 280-04-21. 14
E-mail address: [email protected] 15
16
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
34
ABSTRACT 17
The ability of specific wild Lactococcus (L.) lactis strains to hydrolyze milk proteins to 18
release angiotensin I-converting enzyme (ACE) inhibitory peptides was evaluated. The 19
peptide profiles were obtained from the < 3 kDa water-soluble extract (WSE) and 20
subsequently fractionated by RP-HPLC. The fractions with the lowest IC50 (peptide 21
concentration necessary to inhibit ACE activity by 50%) were L. lactis NRRL B-50571 22
fraction (F)1 (0.034 ± 0.002 μg mL-1
) and L. lactis NRRL B-50572B F1 (0.041± 0.003 23
μg mL-1
). All peptide fractions were analyzed by RP-HPLC-MS/MS. There were 24
identified 21 novel peptide sequences associated to ACE-Inhibitory (ACEI) activity. 25
Several novel ACEI peptides presented peptides encrypted with proven hypotensive 26
activity. In conclusion, specific wild L. lactis strains were able to hydrolyze milk 27
proteins to generate potent ACEI peptides. However, further studies are necessary to 28
find out the relationship between L. lactis strains proteolytic and peptidolytic systems 29
with their ability to biogenerate hypotensive peptides. 30
31
Key Words 32
L. lactis, fermented milk, ACE-Inhibitory peptides 33
34
35
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
35
INTRODUCTION 36
The long-term regulation of blood pressure is associated with the rennin-37
angiotensin system. The conversion of angiotensin I into angiotensin II, a potent 38
vasoconstrictor octapeptide, by the angiotensin-converting enzyme (ACE) [EC 3.4.15.1] 39
has long been known (Skeggs et al., 1956). Hence, the inhibition of this enzyme can 40
reduce high arterial blood pressure through ACE-inhibitory drugs. 41
It is accepted that food proteins may act as precursors of biologically active 42
peptides with different physiological effects. Among these biological activities of 43
peptides, the inhibition of the ACE is one of the most comprehensively studied 44
(Hernández-Ledesma et al., 2005). It has been reported that milk fermentation with 45
highly proteolytic strains of lactic acid bacteria (LAB) is an effective way to increase the 46
amount of bioactive peptides in fermented dairy products (López-Fandiño et al., 2006). 47
Growth of LAB in milk is associated to their proteolytic system to partially degrade 48
caseins and whey proteins to generate free amino acids as well as peptides. These 49
peptides are further hydrolyzed to amino acids by the combined action of an assortment 50
of peptidases (Hugenholtz, 2008). However, for the generation of bioactive peptides, the 51
strains should present a balance between proteolytic activity and the right specificity of 52
the proteinases and peptidases for the generation of ACE-Inhibitory peptides (López-53
Fandiño et al., 2006). 54
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
36
ACE-Inhibitory peptides obtained from the hydrolysis of milk proteins by LAB 55
have shown hypotensive activity (Quirós et al., 2007; Nielsen et al., 2009). Most of the 56
studies have been focused on the ability of lactobacilli strains to biogenerate peptides 57
with ACE-Inhibitory (ACEI) activity (Gobbetti et al., 2000; FitzGerald and Murray, 58
2006). In fact, antihypertensive milk fermented by Lactobacillus helveticus and 59
Saccharomyces cerevisiae (Nakamura et al., 1995) has been commercialized in Japan 60
(Calpis, Calpis Co. Ltd., Tokyo, Japan). On the other hand, LAB such as 61
Enterococcaceae strains have also been evaluated. Similarly, LAB isolated from raw 62
milk were screened and selected on the basis of high ACEI activity (Muguerza et al., 63
2006). Enterococcus faecalis strains stood out as producers of fermented milk with 64
potent ACEI activity. 65
Lactococcus (L.) lactis is one of the LAB most used in the manufacture of 66
fermented dairy products because of their fast lactose fermentation and flavor production 67
(Kuipers, 2001). Native lactococci strains have been associated to the generation of 68
unusual flavors, including higher amounts of certain volatile compounds, than those 69
produced by commercial starter cultures. Moreover, there has been an increased interest 70
in exploring new strains of L. lactis for the improvement of the sensory characteristics of 71
fermented dairy products. In fact, strains of L. lactis isolated from distinct artisanal dairy 72
products presented marked differences in aroma production capacities during their 73
growth in milk (Ayad et al., 1999). On the other hand, Torres-Llanez et al. (2011), 74
recently reported that a wild L. lactis strain presented angiotensin-converting enzyme 75
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
37
activity in Mexican Fresco cheese. Also, specific wild L. lactis strains were explored for 76
their ability to produce ACEI activity in fermented milk (Rodríguez-Figueroa et al., 77
2010). Therefore, native L. lactis strains could not only be excellent aroma producers but 78
also be able to biogenerate ACE-Inhibitory peptides in fermented dairy products. Thus, 79
the objective of this study was to identify and compare the ACE-Inhibitory peptides 80
released from milk proteins through lactic acid fermentation by specific wild L. lactis 81
strains. 82
83
84
MATERIALS AND METHODS 85
Materials 86
Sodium borate, sodium dodecyl sulphate (SDS), 2-mercaptoethanol, ACE (EC 87
3.4.15.1) (5U) which was from rabbit lung powder, Hippurryl-L-histidyl-L-Leucine 88
(Hip-His-Leu) and trifluoroacetic acid were obtained from Sigma Chemical Co. (St. 89
Louis, MO, USA). Acetonitrile was from J.T. Bakers (USA). Lactose, M17 broth and 90
agar were obtained from DIFCO (Sparks, MD, USA). USDA Organic grade A nonfat 91
dry milk was from Organic Valley® (La Farge, WI, USA). 92
93
94
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
38
Strains and growth conditions 95
Two wild L. lactis strains (NRRL B-50571 and NRRL B-50572) obtained from 96
the Dairy Laboratory collection at Centro de Investigación en Alimentación y 97
Desarrollo, A.C (CIAD, Hermosillo, Sonora, Mexico) were deposited in the Agricultural 98
Research service Culture Collection (NRRL), U.S. Department of Agriculture. The 99
strains were propagated in 10 mL of sterile lactose (5 g L-1
) M17 broth and incubated at 100
30°C for 24 h. Fresh cultures were obtained by repeating the same procedure. Initial 101
starter culture were prepared by allowing L. lactis strains to reach 106-10
7 colony-102
forming units (cfu) mL-1
as enumerated on M17 agar containing lactose (5 g L-1
). 103
104
105
Manufacture of fermented milk 106
Reconstituted nonfat dry milk (10%, w/w) was sterilized at 100°C for 20 min. A 107
loop of L. lactis single pre-culture (7-8 log cfu mL-1
) was inoculated into sterilized milk. 108
The inoculated milk was incubated for 12 h at 30°C. Then, cultures were added (3% v/v) 109
to sterilized milk to get the different fermented milk batches. Incubation was carried out 110
at 30°C and stopped at 48 h by pasteurization at 75°C for 1 min. 111
112
113
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
39
Preparation of the Water-Soluble Extracts (WSE) from fermented milk 114
Fermented milk was centrifuged at 20,000 x g (J2-21 rotor, Beckman, USA) for 115
10 min at 0°C. Then, supernatants were collected and ultrafiltered through 3 kDa cut-off 116
membranes (Pall life Sciences, USA) at 9,800 x g for 6 min (J2-21 rotor, Beckman, 117
USA). Permeates were collected, filtered through a 0.45 mm disposable hydrophilic 118
filter and frozen at -80°C until analysis were done. 119
120
Isolation of ACEI peptide fractions by reversed-phase high-performance liquid 121
chromatography (RP-HPLC) 122
Peptide profiles from WSE were obtained by RP-HPLC (1100 series, Agilent 123
Technologies, Japan). Separation was carried out with a Discovery-C18 (250 mm x 4.6 124
mm, 5 μm particle size, 180 Å pore size) column from Supelco (Bellefonte, PA, USA) 125
with a solvent flow rate of 0.25 mL min-1
. Once the column was equilibrated with 126
solvent A (0.04% Trifluoroacetic acid (TFA) in water), 20 μL of the WSE were injected. 127
Peptides were eluted with an increasing gradient of solvent B (0.03% TFA in 128
acetonitrile) from 0% to 45% in solvent A, during 60 min. Peptide profiles monitored at 129
214 nm and 280 nm were collected from five chromatographic runs and freeze-dried to 130
be subjected to ACEI activity analysis and IC50 determination. Peptide fractions (214 131
nm) with the lowest IC50 were eluted once more in order to achieve better separation. 132
This second elution was carried out by using a linear gradient of solvent B (0-15%) in A 133
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
40
during 85 min, with a flow rate of 0.5 mL min-1
. Peptide fractions from this second 134
elution were collected from five chromatographic runs, freeze-dried and stored for 135
further analysis. 136
137
Analysis of peptides by tandem mass spectrometry 138
Mass spectrometry analysis was performed using a 1100 Series LC/MSD Trap 139
(Agilent Technologies, Inc., Waldronn, Germany) equipped with an electro spray 140
ionization source (LC-ESI-MS). The nano column was a C18-300 (150 mm x 0.75 µm, 141
3.5 µm; Agilent Technologies, Inc.) The sample injection volume was 1 µL. Solvent A 142
was a mixture of water-acetonitrile-formic acid (10:90:0.1, v/v/v) and solvent B 143
contained water-acetonitrile-formic acid (97:3:0.1, v/v/v). The gradient was based on the 144
increment of solvent B which was initially set at 3% for 10 min and it took 23 more min 145
to reach 65%. The 0.7 µL min-1
flow rate was directed into the mass spectrometer via an 146
electrospray interface. Nitrogen (99.999%) was used as the nebulizing and drying gas 147
and operated with an estimated helium pressure of 5x10-3
bar. The needle voltage was 148
set at 4 kV. Mass spectra were acquired over a range of 300-2500 mass/charge (m/z). 149
The signal threshold to perform auto MSn analyses was 30,000. The precursor ions were 150
isolated within a range of 4.0 m/z and fragmented with a voltage ramp from 0.35 to 1.1 151
V. Peptide sequences were obtained from mass espectrometry data using the Mascot 152
server (Perkins et al, 1999) through the UniProtKB/Swiss-prot database sequences. 153
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
41
Assay of ACEI activity 154
For ACE-Inhibitory activity analysis of the peptide fractions, the method of 155
Cushman and Cheng (1971) was applied with some modifications. Peptide fractions 156
were dissolved in 1mL of water and the pH was adjusted to 8.3 using 10 N NaOH. The 157
buffered substrate solution was 5 mM Hipurril-L-Histidine-L-Leucine (substrate) in 100 158
mM sodium borate buffer solution containing 300 mM NaCl adjusted to pH 8.3 at 37°C. 159
ACE solution was prepared at 0.1 U mL-1
using distilled water from a milli-QTM
system. 160
Four microtubes were prepared as follows: 161
A =100 μL buffered substrate solution + 40 μL distilled water + 20 μL ACE 162
B =100 μL buffered substrate solution + 20 μL distilled water + 40 μL peptide fraction 163
C =100 μL buffered substrate solution + 40 μL peptide fraction + 20 μL ACE 164
D =100 μL buffered substrate solution + 60 μL distilled water 165
The four microtubes containing the solutions were incubated at 37°C for 35 min. 166
The reaction was stopped adding 250 μL of 1M HCl. Ethyl acetate (1 mL) was added to 167
every sample for the extraction of released hippuric acid. Samples were stirred 168
vigorously for 20 s and centrifuged at 1,500 x g for 10 min. An aliquot of 750 μL of the 169
organic phase was evaporated at 75°C for 30 min. The residue was dissolved in 1 mL of 170
distilled water and stirred vigorously. The absorbance was measured in 400 μL samples 171
at 228 nm. 172
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
42
ACE-Inhibition was calulated as follows: 173
ACE-Inhibitory activity (%) = [ 1- (C - B / A - D) ] X 100 174
ACE-Inhibitory activity can also be measured by the IC50, which is the peptide content 175
(μg mL-1
) neccesary to inhibit ACE activity by 50%. Peptide content (μg mL-1
) in every 176
WSE were determined by Bradford´s method (1976) using a bio-rad protein assay (Bio-177
Rad Laboratories, USA). Bovine serum albumin was used as a standard. The IC50 was 178
calulated using graphical extrapolation by plotting ACE inhibition as a function of 179
peptide content (Donkor et al., 2007). Therefore, every single sample was adjusted at 180
least to three levels of known peptide concentration (μg mL-1
) by standard volume 181
dilution. Measurements were taken in duplicates. 182
183
Statistical analysis 184
Experiments were carried out in triplicates and normality of data was evaluated 185
as a prerequisit before one way ANOVA analysis was performed. Differences between 186
means were assesed by Tuckey-Kramer multiple-comparison test and were considered 187
significant when P < 0.01. Statistical analysis was performed by using NCSS 2007 188
software (Kaysville, UT, USA). 189
190
191
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
43
RESULTS AND DISCUSSION 192
Peptide fraction profiles from milk fermented by specific wild L. lactis strains 193
Figure 1 shows WSE peptide fraction profiles produced by specific wild L. lactis strains 194
monitored at 214 nm absorbance. Unfermented milk was used as a control. The area 195
under the curve of each peptide profile was evaluated as an indirect measure of the grade 196
of proteolysis. Results showed significant differences (P < 0.01) between fermented milk 197
peptide profiles and the control. On the other hand, the peptide profiles obtained from 198
milk fermented with different strains of L. lactis were similar. The first peak eluted after 199
12 min in all samples. The most notorious amount of peptides eluted between 12 and 25 200
min when the concentration of acetonitrile was between 9 - 13.5%, which may be related 201
to the hydrophobic nature of the eluted peptide. It has been suggested that there is a 202
close relationship between hidrophobicity and positively charged amino acids in the C-203
terminal position and ACE-Inhibitory peptides derived from milk proteins (Pripp et al., 204
2004). Thus it is very likely that peptides eluting in the first 25 minutes present ACEI 205
activity. 206
On the other hand, when WSE were monitored at 280 nm, only three peaks eluted 207
between 16 and 20 min (figure not shown). These peptides may have ACEI activity 208
since they were of aromatic nature. Jianping et al. (2006) reported the relevant presence 209
of aromatic amino acids in the ACEI peptides structure. 210
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
44
A comparison of L. lactis strains ability for hydrolyzing milk proteins was shown 211
by recording area counts from the peptide chromatographic profiles (Figure 2). Even 212
though, there was not a significant difference (P > 0.01), milk fermented by L. lactis 213
NRRL B-50571 presented lower degree of proteolysis than NRRL B-50572. In general, 214
L. lactis strains have a complex proteolytic system which is able to hydrolyze milk 215
proteins to amino acids or peptides essential for growth (Savijoki et al., 2006). Thus, 216
milk proteins proteolysis should be a prerequisite to find out peptides with bioactivity. 217
On the other side, Pripp et al.(2004), established a relationship between milk derived 218
peptides and their ACEI activity through quantitative structure-activity relationship 219
model. Therefore, it was necessary to identify the structure of peptides associated to the 220
bioactivity. 221
222
Peptide fractions with angiotensin I-converting enzyme inhibitory activity 223
Peptide chromatographic profiles were divided into 6 fractions and collected for 224
further evaluation. Peptide profiles obtained at 214 nm were divided into F1-F5 225
fractions, meanwhile peptide profiles obtained at 280 nm corresponded to F6. Peptide 226
fractions F1-F6 showed remarkable IC50 with values ranging from 0.034 ± 0.002 to 0.61 227
± 0.19 μg mL-1
(Figure 3). Results did not show significant difference (P > 0.01) 228
between all peptide fractions IC50. However, the peptide fractions IC50 obtained from 229
milk fermented by L. lactis strains NRRL B-50571 (0.076 ± 0.004 and 0.034 ± 0.002 μg 230
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
45
mL-1
for F1 and F6, respectively) and milk fermented by L. lactis NRRL B-50572 231
(0.041± 0.003 and 0.084 ± 0.003 μg mL-1
for F1 and F2, respectively) showed the lowest 232
values (figure 3). Quirós et al. (2007) reported a < 3 kDa sub-fractionated water-soluble 233
extract with an IC50 of 0.8 μg mL-1
released by the hydrolytic action of Enterococcus 234
faecalis on skimmed milk during 48 h fermentation time. Moreover, Lactobacillus 235
helveticus and Saccharomyces cerevisiae were able to hydrolyze skimmed milk 236
obtaining two different peptides with the minimum IC50, 2.8 and 1.6 μg mL-1
(Nakamura 237
et al., 1995). Therefore, the results suggest that the specific wild L. lactis strains of this 238
study have remarkable ACE-Inhibitory activity. 239
240
Identification of peptides with ACEI activity derived from L. lactis fermented milk 241
Peptides associated to every fraction were identified by tandem mass 242
spectrometry. Twenty-one new peptides associated to angiotensin I-converting enzyme 243
inhibitory activity were identified and their molecular weight was calculated (Table 1). 244
The only peptide already reported was LHLPLPL, which was found in milk fermented 245
by Enterococcus faecalis (Quirós et al., 2007). The presence of the peptide sequence 246
TVQVTSTAV in milk fermented by specific wild L. lactis strains suggested that L. 247
lactis NRRL B-50571 and NRRL B-50572 strains may present similar proteolytic or 248
peptidolytic systems. On the other hand, only one peptide sequence presented Pro in the 249
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
46
C-terminal position, therefore, these results may suggest the absence of Proline-specific 250
peptidases such as Pep I, Pep R and Pep X. 251
The peptide sequence with the lowest molecular weight (505 Da) was AESIS 252
derived from αS1-CN (f62-65). On the other hand, the longest sequence obtained 253
presented 21 amino acids with a molecular weight of 2048 Da derived from 254
serotransferrin (f506-526). It has been suggested that ACEI peptides usually include 2-255
12 amino acids, however it has been reported that peptides of up to 27 amino acids may 256
present ACEI. Another key point is the ability of C-terminal hydrophobic amino acids 257
like Pro to bind ACE (López-Fandiño et al., 2006). In fact, F1 obtained from milk 258
fermented with L. lactis NRRL B-50571 presented the peptide sequence 259
HPHPHLSFMAIPP with Pro in the C-terminal position. Pripp et al. (2004), also 260
specified a relationship between hydrophobicity and positively charged amino acid in 261
the C-terminal position and ACEI activity. Indeed, the peptide sequence DDQNPH, 262
which also was presented in F1 presented the positively charged residue histidine in the 263
C-terminal position. Both of these peptide sequences presented in F1 produced by L. 264
lactis NRRL B-50571 fermented milk presented the lowest IC50. 265
It has been reported that α, β and κ caseins are precursors of bioactive peptides 266
(Mills et al., 2011). However in this work it was found that casein proteins as well as 267
whey proteins, such as β-Lactoglobulin and α-Lactalbumin, may be relevant sources of 268
peptides with ACEI activity. 269
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
47
Milk fermented by L. lactis NRRL B-50571 showed HPHPHLSFMAIPP 270
derived from κ-CN (f98-110) and SLPQNIPPL derived from β-CN (f69-77) which have 271
encrypted the hypotensive tripeptide (IPP) reported by Nakamura et al., (1995). 272
Additionally, milk fermented by L. lactis NRRL B-50572 showed 273
QEPVLGPVRGPFPIIV derived from β-CN (f194-209). Thus, this amino acid sequence 274
included the peptide VLGPVRGPFP which was reported by Quirós et al. (2007). 275
Finally, the peptide fragment YPSYGL obtained from κ-CN (f35-40) found in 276
fermented milk by L. lactis NRRL B-50571 and NRRL B-50572 strains showed the 277
dipeptide YP reported before (Yamamoto et al., 1999). 278
279
CONCLUSIONS 280
The peptide profiles of the distinct fractions obtained from the hydrolysis of milk 281
proteins by specific wild L. lactis strains were similar. Nevertheless, there were 282
differences in the degree of proteolysis that may be related to the action of specific 283
peptidases and proteinases of each L. lactis strain. Moreover, this research suggests that 284
the degree of proteolysis may be a prerequisite to ACEI activity, however it seems to be 285
that differences in L. lactis strains proteolytic and peptidolytic systems determine the 286
peptide sequences associated to ACE inhibition. Therefore, studies are under way to 287
determine enzymatic activity differences among specific wild L. lactis strains. 288
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
48
Specific wild L. lactis strains were able to release twenty-one new encrypted 289
milk peptides with potent ACE-Inhibitory activity through a fermentation process not 290
only from caseins but also from whey proteins. 291
292
ACKNOWLEDGEMENT 293
This study was supported by the Mexican National Council of Science and 294
Technology (CONACYT) research grant No. 42340 Z. We would like to thank to 295
Carmen Estrada MD for technical support. 296
297
REFERENCES 298
Ayad, E.H.E., Verheul, A., Jong, C., Wouters, J.T.M., & Smit, G. 1999. Flavour 299
forming abilities and amino acid requirements of Lactococcus lactis strains 300
isolated from artisanal and non-dairy origin. Int. Dairy J., 9: 725-735. 301
Barrett, E., Hayes, M., Fitzgerald, G.F., Hill, C., Stanton, C., & Ross, R.P. 2005. 302
Fermentation, cell factories and bioactive peptides: food grade bacteria for303
production of biogenic compounds. Australian J. Dairy Technol., 60:157-162. 304
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of305
microgramquantities of protein utilizing the principle of protein-dye binding.306
Anal. Biochem., 72: 248-254. 307
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
49
Cushman, D.W., & Cheung, H.S. 1971. Spectrophotometric assay and properties of the 308
angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol., 20: 1637-309
48. 310
Donkor, O.N., Henriksson, A., Singh, T.K., Vasiljevic, T., & Shaha, N.P. 2007. ACE-311
inhibitory activity of probiotic yoghurt. Int. Dairy J., 17:1321-1331. 312
FitzGerald, R., & Murray, B. 2006. Bioactive peptides and lactic fermentation. Int J. 313
Dairy Technol., 59: 118-125. 314
Fox, P.F., McSweeney, P.L.H. 2003. Advnced dairy chemistry. Proteins. 3rd
Edition. 315
Vol. 1. Part A.Kluwer Academic/Plenum Publishers. NY, USA. Pp 161, 163, 316
165, 166. 317
Gobbetti, M., Ferranti, P., Smacchi, E., Goffredi, F., & Addeo, F. 2000. Production 318
ofangiotensin-I-converting-enzyme-inhibitory peptides in fermented milks 319
started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus 320
lactis subsp. cremoris FT4. Appl. Environ Microbiol., 66: 3898-904. 321
Gutiérrez-Méndez, N., Vallejo-Cordoba, B., González-Córdova, A.F., Nevárez- 322
Moorillón, G.V., & Rivera-Chavira, B. 2008. Evaluation of aroma generation of 323
Lactococcus lactis with an electronic nose and sensory analysis. J. Dairy Sci., 324
91:49-57. 325
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
50
Hernández-Ledesma, B., Miralles, B., Amigo, L., Ramos, M., & Recio, I. 2005. 326
Identification of antioxidant and ACE-inhibitory peptides in fermented milk. J. 327
Sci. Food Agri., 85:1041-1048. 328
Hugenholtz, J. 2008. The lactic acid bacterium as a cell factory for food ingredient 329
production. Int. Dairy J., 18:466-475. 330
Jianping, W., Rotimi, A., & Shuryo N. 2006. Structural requirements of angiotensin I- 331
converting enzyme inhibitory peptides: Quantitative structure-activity 332
relationship modeling of peptides containing 4-10 amino acid residues. QSAR 333
Comb. Sci., 25:873-880. 334
Kuipers, O.P. 2001. Complete DNA sequence of Lactococcus lactis adds flavor to 335
Geno- mics. Genome Res., 11: 673-674. 336
López-Fandiño, R., Otte, J., & van Camp, J. 2006. Physiological, chemical technological 337
aspects of milk-protein-derived peptides with antihypertensive and ACE-338
inhibitory activity. Int. Dairy J., 16:1277-1293. 339
Mills, S., Ross, R.P., Hill, C., FitzGerald, G.F., & Stanton, C. 2011. Milk intelligence: 340
Mining milk for bioactive substances associated with human health. Int. Dairy J., 341
21:377-401. 342
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
51
Muguerza, B., Ramos, M., Sánchez, E., Manso, M.A., Miguel, M., Aleixandre, A., 343
Delgado, M.A., & Recio, I. 2006. Antihypertensive activity of milk fermented 344
by Enterococcus faecalis strains isolated from raw milk. Int. Dairy J., 16:61-69. 345
Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Vamazak, S., & Takano, T. 1995. 346
Purification and characterization of angiotensin I-converting enzyme inhibitors 347
from sour milk. J. Dairy Sci., 78:777-783. 348
Nielsen, M.S., Martinussen, T., Flambard, B., Sørensen, K., & Otte, J. 2009. Peptide 349
profiles and angiotensin-I-converting enzyme inhibitory activity of fermented 350
milk products: Effects of bacterial strain, fermentation pH, and storage time. Int. 351
Dairy J., 19:155-165. 352
Perkins, D.N., Pappin D.J.C., Creasy, D.M., & Cottrell, J.S. 1999. Probablility-based 353
protein identification by searching sequence databases using mass spectrometry 354
data. Electrophoresis, 20: 551-3567. 355
Pripp, A., Isaksson, T., Stepaniak, L., & Sørhaug, T. 2004. Quantitative structure-356
activity relationship modeling of ACE-inhibitory peptides derived from milk 357
proteins. Eur. Food Res. Technol., 219: 579-583. 358
Quirós, A., Ramos, M., Muguerza, B., Delgado, M., Miguel, M., Alixandre, A., & 359
Recio, I. 2007. Identification of novel antihypernensive peptides in milk 360
fermented with Enterococcus faecalis. Int. Dairy J., 17: 33-41. 361
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
52
Rodríguez-Figueroa, J.C., Reyes-Díaz, R., González-Córdova, A.F., Troncoso-Rojas, R., 362
Vargas-Arispuro, I., Vallejo-Cordoba, B. 2010. Angiotensin-converting enzyme 363
inhibitory activity of milk fermented by wild and industrial Lactococcus lactis 364
strains. J. Dairy Sci., 93: 5032-5038. 365
Savijoki, K., Ingmer, H., & Varmanen, P. 2006. Proteolytic systems of lactic acid 366
bacteria. Appl. Microbiol. Biotechnol., 71:394-406. 367
Skeggs, L.T., Kahn, J.R., & Shumway, N.P. 1956. The preparation and function of the 368
hypertension-converting enzyme. J. Exp. Med., 103: 295-299. 369
Torres-Llanez, M.J., González-Córdova, A.F., Hernández-Mendoza, A., García, H.S., & 370
Vallejo-Cordoba, B. 2011. Angiotensin-converting enzyme inhibitory activity in 371
Mexican Fresco cheese. J. Dairy Sci., 94: 3794-3800. 372
Van Huynegem, K., Loos, M., & Steidler, L. 2009. Immunomodulation by genetically 373
engineered lactic acid bacteria. Frontiers in Bioscience, 14:4825-4835. 374
Yamamoto, N., Maeno, M., & Takano, T. 1999. Purification and characterization of an 375
antihypertensive peptide from a yogurt-like product fermented by Lactobacillus 376
helveticus CPN4. J. Dairy Sci., 82:1388-1393. 377
378
379
380
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Table 1. Identification of peptides obtained from milk fermented by specific wild L. 381
lactis strains associated to ACEI activity. 382 383
Samplea Experimental Theorical Molecular ion Protein fragment Sequence
Mass Mass (m/z) selected for
MS/MS (charge)
NRRL B- 723.9 724.3 362.9(+2) α-La (f63-68) DDQNPH
50571 1032.8 1033.5 517.4 (+2) α-La (f82-89) LDDDLTDDI
F1 698.6 698.3 350.3 (+2) κ-CN (f35-40) YPSYGL
1479.0 1479.7 740.5 (+2) κ-CN (f98-110) HPHPHLSFMAIPP
1035.7 1035.5 518.8 (+2) α-La (f55-62) YDTQAIVQ
1386.8 1387.7 462.3 (+3) α-La (f100-111) DDDLTDDIMCV
585.9 585.2 586.7 (+1) κ-CN (f35-39) YPSYG
F2 505.9 585.2 506.9 (+1) αS1-CN (f62-66) AESIS
F3 830.1 830.5 416.1 (+2) β-CN (f22-28) SITRINK
1051.4 1051.5 526.7 (+2) αS1-CN (f80-88) HIQKEDVPS
904.1 904.5 453.0 (+2) κ-CN (f161-169) TVQVTSTAV
F4 904.3 904.5 453.2 (+2) κ-CN (f161-169) TVQVTSTAV
1038.4 1038.6 520.2 (+2) αS2-CN (f115-124) NAVPITPTLN
977.1 977.6 489.6 (+2) β-CN (f69-77) SLPQNIPPL
F5 1716.9 1717.0 859.4 (+2) β-CN (f194-209) QEPVLGPVRGPFPIIV
1150.4 1150.7 576.2 (+2) β-CN (f199-209) GPVRGPFPIIV
977.2 977.6 489.7 (+2) β-CN (f69-77) SLPQNIPPL
1094.4 1094.6 548.2 (+2) κ-CN (f25-33) YIPIQYVLS
F6 904.4 904.5 453.5 (+2) κ-CN (f161-169) TVQVTSTAV
1356.7 1357.7 453.2 (+3) κ-CN (f157-169) PEINTVQVTSTAV
591.8 592.3 198.3 (+3) Serotransferrin (f448-453) GYLAVA
NRRL B- 1371.53 1372.7 686.6 (+2) β-CN (f129-140) DVENLHLPLPLL
50572 698.6 698.3 350.2 (+2) β-CN (f35-40) YPSYGL
F1 549.8 550.2 550.9 (+1) β-Lg (f60-64) ENGEC
F2 904.2 904.5 453.1 (+2) κ-CN (f161-169) TVQVTSTAV
F3 904.2 904.5 453.1 (+2) κ-CN (f161-169) TVQVTSTAV
F5 1150.5 1150.7 576.3 (+2) β-CN (f199-209) GPVRGPFPIIV
F6 922.4 922.4 922.4 (+1) α-La (f86-93) TDDIMCVK a = Fractions collected from milk fermented by L. lactis NRRL B-50571 and NRRL B-50572. 384
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
54
Figure captions 385
Figure 1. Peptide profiles corresponding to the WSE < 3 kDa fraction obtained from the 386
fermentation of milk by specific wild L. lactis strains by RP-HPLC analysis at an 387
absorbance of 214nm. NRRL B-50571 and NRRL B-50572 = Specific wild L. lactis 388
strains. C = unfermented milk. 389
Figure 2. Proteolysis degree of the different peptide profiles through the area under the 390
curve corresponding to the WSE < 3 kDa fraction obtained from the fermentation of 391
milk by specific wild L. lactis strains by RP-HPLC analysis at an absorbance at 214nm 392
and 280 nm. Unfermented milk was used as control. aMean values ± SD ( n = 4). 393
Figure 3. IC50 values of the peptide fractions obtained by hydrolysis of milk proteins by 394
specific wild L. lactis strains obtained by RP-HPLC. IC50, represents the concentration 395
of the peptide fraction (μg mL-1
) necessary to inhibit ACE activity at 50%. Data 396
represented means values ± SD (n = 3). Statistical differences were considered with P ˂ 397
0.01, using one way ANOVA and Tuckey-Kramer test as multiple-compare test. F = 398
Peptide fraction. F1-F5 = obtained at 214 nm; F6 = obtained at 280 nm. 399
Figure 4. Mass spectrum corresponding to a peptide sequence collected from the WSE 400
F1 obtained from milk fermented by L. lactis NRRL B-50571. (A) Double-charged ion 401
362.9 m/z.;(B) MS/MS Spectrum for the specified ion in (A). After interpretation and 402
comparison in database, the fragment amino acid sequence matched with α-La (f63-68). 403
404
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
55
Figure 1 405
406
Minutes 407
408
Figure 2 409
410
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
56
411
Figure 3 412
413
414
Figure 4 415
416
MILK FERMENTED BY L. LACTIS WITH ACEI ACTIVITY
57
417
Capítulo 4
Specific wild Lactococcus lactis strains able to
ferment milk with relevant blood pressure and
heart rate lowering effect
Artículo enviado al: British Journal of Nutrition
59
60
L. lactis fermented milk hypotensive effect
61
Milk fermented by specific Lactococcus lactis strains with relevant 1
blood pressure and heart rate lowering effect 2
3 J.C. Rodríguez-Figueroa, A.F. González-Córdova, H. Astiazaran-García, B. 4
Vallejo-Cordoba* 5 6
Centro de Investigación en Alimentación y Desarrollo A.C. (CIAD, A.C.). Carretera a la 7
Victoria Km. 0.6, 83304, Hermosillo, México 8
* Corresponding Author. 9
Belinda Vallejo-Cordoba 10 Centro de Investigación en Alimentación y Desarrollo A.C. (CIAD, A.C.). Carretera a la 11 Victoria Km. 0.6, 83304, Hermosillo, México 12
Phone: +52 (662) 289- 24-00 ext. 303; Fax: +52 (662) 280- 04-21. 13 E-mail address: [email protected] 14
15
Milk fermented by specific Lactococcus lactis strains, Spontaneously hypertensive rats 16
(SHR) as animal model, blood pressure and heart rate lowering effect 17
Footnotes 18
Abbreviations: L. lactis, Lactococcus lactis; ACE, angiotensin I-converting enzyme; 19
SHR, spontaneously hypertensive rats; BW, body weight; SBP, systolic blood pressure; 20
DBP, diastolic blood pressure; HR, heart rate; PP, pulse pressure; PWV, pulse wave 21
velocity; LAB, Lactic acid bacteria; CIAD, A.C., Centro de Investigación en 22
Alimentación y Desarrollo A.C.; cfu, colony-forming units; USDA, United States 23
Department of Agriculture; AOAC, Official Methods of Analysis; EPA, Environmental 24
Protection Agency; LSD, Least significant difference; SEM, mean standard error; NRRL 25
B-50571-3, milk fermented by L. lactis NRRL B-50571 (35 mg protein/kg BW); NRRL 26
B-50572-3, milk fermented by L. lactis NRRL B-50572 (35 mg protein/kg BW); NRRL 27
B-50571-5 milk fermented by L. lactis NRRL B-50571 (50 mg protein/kg BW); NRRL 28
B-50572-5 milk fermented by L. lactis NRRL B-50572 (50 mg protein/kg BW); ND, not 29
detected; Vs, versus. 30
L. lactis fermented milk hypotensive effect
62
31
Abstract 32
Previous studies demonstrated that milk fermented by specific Lactococcus (L.) lactis 33
strains significantly inhibit the activity of angiotensin I-converting enzyme (ACEI). 34
However, to date there is not a clear relationship between ACEI and antihypertensive 35
effects in animal models. Therefore, the aim of the present research was to investigate 36
the antihypertensive and heart rate (HR) lowering effect of milk fermented by specific L. 37
lactis in a murine model. Spontaneously hypertensive male rats (SHR) (271 ± 14 g) were 38
randomized into four treatment groups: oral administration of milk fermented by L. 39
lactis NRRL B-50571 or L. lactis NRRL B-50572 at 35 or 50 mg protein /kg of body 40
weight (BW). Two more groups were fed with different solutions as controls: a saline 41
solution was the negative control, meanwhile CaptoprilTM
(40 mg/kg BW), a proven 42
ACE inhibitor was the positive control. Blood pressure and heart rate were monitored by 43
the tail cuff method before treatments and 2, 4, 6 and 24 h post oral administration. 44
Results demonstrated that milk fermented by L. lactis NRRL B-50571 as well as milk 45
fermented by L. lactis NRRL B-50572 presented an important systolic (SBP) and 46
diastolic blood pressure (DBP) and HR lowering effect. Thus, milk fermented by 47
specific L. lactis strains may present potential benefits in the prevention and treatment of 48
cardiovascular diseases associated to hypertension in humans. 49
50
Key Words: 51
Lactococcus lactis; Fermented milk; Antihypertensive effect; Functional food; 52
Spontaneously hypertensive rat 53
54
55
L. lactis fermented milk hypotensive effect
63
56
Hypertension has become a serious health problem, which has been considered an 57
important cardiovascular disease risk factor, especially in developing countries (Anadón 58
et al., 2010). The long-term regulation of blood pressure is associated with the rennin-59
angiotensin system. The conversion of angiotensin I into angiotensin II, a potent 60
vasoconstrictor octapeptide, by the angiotensin-converting enzyme (ACE) [EC 3.4.15.1] 61
has long been known (Skeggs et al., 1956). Hence, the inhibition of this enzyme can 62
reduce high arterial blood pressure through ACE-inhibitory compounds. 63
Blood pressure is monitored by the systolic blood pressure (SBP) and diastolic 64
blood pressure (DBP). Brachial SBP is overall the best predictor of future cardiovascular 65
risk for the entire hypertensive population. However, DBP must be measured in order to 66
calculate pulse pressure (PP), which has become a surrogate marker of central elastic 67
large artery stiffness and a useful predictor of cardiovascular risk in the elderly 68
population (Stanley 2007; Safar et al., 2003). On the other side, HR is an important 69
determinant of myocardial oxygen consumption and of cardiac work. Several 70
experimental lines of research consider HR as an important risk factor for 71
atherosclerosis. In fact, heart rate reduction may represent an important strategy for the 72
treatment of patients with a wide range of cardiac disorders (Palatini, 2009). Therefore, 73
SBP, DBP and heart rate (HR) measurements give a wide view related to cardiovascular 74
disorders. Because of the high prevalence of hypertension and serious health 75
consequences, lifestyle modifications, including dietary interventions, are recommended 76
to help prevent and treat hypertension (Chobanian et al., 2003). Research with 77
hypertensive animals (Muguerza et al., 2006) and humans (Aihara et al., 2005) indicate 78
that milk peptides derived from casein and whey may have a hypotensive effect. 79
Milk proteins have received increased attention as potential ingredients in health-80
promoting functional foods. It is accepted that proteins from milk may act as precursors 81
of biologically active peptides with different physiological effects on the digestive, 82
L. lactis fermented milk hypotensive effect
64
endocrine, cardiovascular, immune and nervous systems (Korhonen 2009). Indeed, it has 83
been reported that an effective way to increase the amount of bioactive peptides in dairy 84
products is by milk fermentation with highly proteolytic strains of lactic acid bacteria 85
(LAB) (López-Fandiño et al., 2006). LAB growth in milk is dependent on the specific 86
proteolytic and peptidolytic systems for the generation of free amino acids and free 87
peptides as a source of nitrogen (Hugenholtz, 2008). In fact, several studies suggested 88
that peptides released by Enterococcus faecalis strains from milk proteins were able to 89
decrease arterial blood pressure in spontaneously hypertensive rats (SHR) (Muguerza et 90
al., 2006; Quirós et al., 2007). 91
L. lactis is one of the most well studied LAB because of its importance as part of 92
commercial starter cultures used in the manufacture of fermented dairy products 93
(Odamaki et al., 2011). It has been reported that L. lactis strains are able to improve the 94
organoleptic characteristics of dairy products (Ayad 2009). Previous studies in our 95
laboratory showed that specific L. lactis strains isolated from native ecosystems were 96
able to produce remarkable aroma profiles in fermented milk (Gutiérrez-Méndez et al., 97
2008). In addition, fermented milks with these specific strains were able to inhibit ACE 98
activity. However, the antihypertensive effects of fermented milk with these specific L. 99
lactis strains has not been tested. Therefore, the objective of this study was to evaluate 100
the blood pressure and HR lowering effect of milk fermented by specific L. lactis 101
strains in an animal model. 102
103
104
105
106
107
108
L. lactis fermented milk hypotensive effect
65
Materials and methods 109
Strains and growth conditions 110
L. lactis strains were deposited at the Agricultural Research Service Culture 111
Collection (NRRL) of the National Center for Agricultural Utilization Research (U.S. 112
Department of Agriculture Peoria, ILL) . Strains were routinely propagated in 10 ml of 113
sterile lactose (5 g/l) M17 broth (DIFCO Sparks, MD, USA) and incubated at 30°C for 114
24 h. Fresh cultures were obtained by repeating the same procedure. The initial starter 115
culture of each L. lactis strain reached 106-10
7 colony-forming units (cfu)/ ml
as 116
enumerated on M17 agar supplemented with lactose (5 g/l). 117
Manufacture of fermented milk 118
Organic grade A nonfat dry milk from Organic Valley® (La Farge, WI, USA) 119
was reconstituted in purified water at 10% (w/w) and sterilized (100°C, 20 min). Every 120
single specific L. lactis strain was inoculated with a loop in sterilized milk with an initial 121
bacterial population of 7-8 log cfu/ml as pre-cultures. The inoculated milk was incubated 122
for 12 h at 30°C. Pre-cultures were added (3% v/v) to sterilized milk to get the different 123
fermented milk batches. Incubation was performed at 30°C and it was stopped at 48 h by 124
pasteurization at 75°C for 1 min for obtaining samples for the single doses bioassay. 125
Sample preparation 126
Samples of specific L. lactis fermented milk for the single doses bioassay were 127
obtained by centrifugation at 20,000 x g (J2-21 rotor, Beckman, USA) for 10 min at 128
0°C. The supernatants were collected and lyophilized with a freeze dryer (Labconco, 129
USA). Total protein (Method 960.52 AOAC, 1998), calcium, magnesium and potassium 130
(EPA 3052), total fat (Method 942.05 AOAC, 1998) and lactose (Method 930.28 131
AOAC, 1998) contents of lyophilized whey were evaluated (Table 1). 132
133
L. lactis fermented milk hypotensive effect
66
Table 1. Chemical composition of the whey fractions obtained from milk fermented by specific L. lactis strains
(Mean values with their standard deviation)
NRRL
B-50571-3
NRRL
B-50572-3
NRRL
B-50571-5
NRRL
B-50572-5
Mean SD Mean SD Mean SD Mean SD
Protein (mg/d) 35 3
35 3
50 3
50 3
Fat (g/d) ND
ND
ND
ND
Lactose (mg/d) 1250 30
1310 20
1790 43
1870 28
Ca (mg/d) 3.7 0.01
3.6 0.03
5.3 0.02
5.23 0.04
K (mg/d) 4.4 0.1
4.3 0.1
6.3 0.2
6.2 0.1
Mg (mg/d) 0.3 0.01 0.3 0.03 0.5 0.02 0.4 0.1
NRRL B-50571-3, milk fermented by L. lactis NRRL B-50571 (35 mg protein/kg BW); NRRL B-50572-3, milk fermented by L. lactis NRRL B-50572 (35 mg protein/kg BW); NRRL B-50571-5, milk fermented by L. lactis NRRL B-50571 (50 mg protein/kg BW); NRRL
B-50572-5, milk fermented by L. lactis NRRL B-50572 (50 mg protein/kg BW). d = dosage, ND = Not detected
Experimental protocol with SHR 134
135
Forty-two male SHR (4-5 weeks old, 72±7 g BW) were obtained from Harlan 136
Laboratories, INC, (Indianapolis, IL, USA). SHR were weaned for five weeks and 137
conditioned for arterial blood pressure monitoring. Rats were randomly housed in pairs 138
per cage at 21 ± 2°C with 12 h light/dark cycles, 52 ± 6 % relative humidity and with ad 139
libitum intake of a standard diet (Teklad, Harlan Laboratories, USA) and purified water. 140
SHR (12-13 weeks old, 271±14 g BW) were divided into six groups of seven rats (n = 141
7): Oral administration of saline solution was the negative control, meanwhile 142
CaptoprilTM
(proven hypotensive drug) (40 mg/kg body weight (BW)) was the positive 143
control. On the other hand, lyophilized whey fractions of milk fermented by L. lactis 144
NRRL B-50572 or NRRL B-50571 were dissolved in saline solution. Treatments were 145
NRRL B-50572-3 (35 mg protein/kg BW), NRRL B-50572-5 (50 mg protein/kg BW), 146
NRRL B-50571-3 (35 mg protein/kg BW) and NRRL B-50571-5 (50 mg protein/kg 147
BW). 148
149
Conscious SHR received a single dose through a canula between 8:30 and 9:30 150
am to eliminate circadian cycles. Animals were restrained in the warming chamber for 151
L. lactis fermented milk hypotensive effect
67
20 min at 32°C to detect pulsations through the caudal artery. Systolic (SBP) and 152
diastolic (DBP) blood pressures as well as heart rate (HR) were taken five times before 153
administration at 2, 4, 6 and 24 h post-administration. Measurements were obtained 154
using the non-invasive blood pressure system included photoelectric sensor, amplifier, 155
automatic inflation cuff and software (Model 229, IITC, USA). The animal experimental 156
procedures were done following the guidelines and supervision of the CIAD, A.C. 157
Committee of Ethics for scientific research. 158
159
160
Statistical analysis 161
Data normality was evaluated as a prerequisit before one way analysis of 162
variance was carried out. Differences among means were assesed by the Fisher´s LSD 163
multiple-comparation test and they were considered significant when P < 0.05. Data 164
were processed by the NCSS 2007 statistical program. 165
166
Results 167
168
Antihypertensive effects of milk fermented by specific L. lactis strains 169
170
Figure 1 shows that after 7 weeks, SHR rats became hypertensive. Rats presented 171
more than 150 mm Hg systolic blood pressure for more than 4 weeks which is a 172
prerequisite for being considered hypertensive (Okamoto Kozo and Aoki Kyuzo, 1963). 173
174
L. lactis fermented milk hypotensive effect
68
175
Figure 1. Development of hypertension through age. Systolic blood pressure = SBP. (Values are means 176
with their standard error) (n= 42). 177
178
SBP changes are shown in figure 2a. Results showed the maximal SBP 179
reductions at 6 h post oral administration. SHR treated with the whey fractions of milk 180
fermented by L. lactis NRRL B-50572-5 and L. lactis NRRL B-50571-3 presented the 181
more relevant decrement of SBP, 16.7 ± 3.5 mm Hg and 17.7 ± 4.0 mm Hg, 182
respectively, although treatments were not significantly different (P ˂ 0.05). 183
184
The maximum decrease at 6h was observed in animals treated with CaptoprilTM
185
which was significantly different from the treatments (P ˂ 0.05). However, the SBP 186
measurements 24 h post administration showed that SHR treated with the whey fraction 187
of milk fermented by L. lactis NRRL B-50572-5 presented 4.3 mm Hg less than rats that 188
were treated with CaptoprilTM
. These results suggests that L. lactis NRRL B-50572-5 189
fermented milk may have an important residual blood pressure reducing effect. 190
Moreover, a remarkable 15.3 mm Hg SBP decrement between SHR that received the 191
whey fraction of milk fermented by L. lactis NRRL B-50572-5 and SHR treated with 192
saline was found. Hence, blood pressure measurements suggested an absence of dosage 193
dependent relationship between the protein content of the whey fraction corresponding 194
to milk fermented by L. lactis NRRL B-50571 and its ability to reduce SBP, meanwhile 195
L. lactis fermented milk hypotensive effect
69
the whey fraction of milk fermented with L. lactis NRRL B-50572 was dosage 196
dependent. 197
198
Table 2. Measurements of systolic blood pressure (SBP) and diastolic blood pressure (DBP) in spontaneously hypertensive rats
(SHR) treated with milk fermented by specific L. lactis strains at different times (Mean values with their standard error)
2 h
4 h
6 h
24 h
SBP
(mm Hg)
DBP
(mm Hg)
SBP
(mm Hg)
DBP
(mm Hg)
SBP
(mm Hg)
DBP
(mm Hg)
SBP
(mm Hg)
DBP
(mm Hg)
M
Mean
S
SEM
M
Mean
S
SEM
M
Mean
S
SEM
M
Mean
S
SEM
M
Mean
S
SEM
M
Mean
S
SEM
M
Mean
S
SEM
M
Mean
S
SEM
Saline 1
184.3 2
2.8
1143.8
99.1
1193.7
88.7
1152
33.6
1193.3
22.1
1148.6
88.5
2203.9
22.2
1154.9
22.0
CaptoprilTM
1
177.4
1
13
1
137.5
5
5.8
1
173.5
7
7.1
1
129.4
9
9.7
1
156.5**
5
5.7
1
119.3*
5
5.8
1
192.9
5
5.8
1
152.2
8
8.6
NRRL
B-50572-3
1
184.5
1
11
1
153.4
8
8.4
1
193.3
1
12
1
141.3
1
14
1
187.2‡‡
9
9.6
1
142.3‡
8
8.7
1
193.0
9
9.6
1
154.4
9
9.6
NRRL
B-50572-5
1
178.5
1
11
1
141.4
9
9.4
1
184.8
7
7.2
1
131.0
7
7.5
1
176.6*‡‡
3
3.5
1
125.0*
9
9.4
1
188.6
3
3.5
1
150.8
5
5.2
NRRL
B-50571-3
1
196.2
5
5.9
1
163.4‡
1
11
1
195.5‡
4
4.9
1
150.1
4
4.7
1
175.6*
4
4.2
1
137.9
1
11
1
195.8
4
4.1
1
165.1
6
6.1
NRRL B-50571-5
1195.2
22.9
1159.1
77.1
1186.0
99.7
1141.1
110
1181.9‡‡
44.3
1132.6
77.1
1197.5
44.4
1161.5
88.6
199 NRRL B-50572-3, milk fermented by L. lactis NRRL B-50572 (35 mg protein/kg BW); NRRL B-50572-5, milk fermented by L. lactis 200 NRRL B-50572 (50 mg protein/kg BW); NRRL B-50571-3, milk fermented by L. lactis NRRL B-50571 (35 mg protein/kg BW); NRRL B-201 50571-5, milk fermented by L. lactis NRRL B-50571 (50 mg protein/kg BW). * P ˂ 0.05 vs Saline, ** P˂ 0.01 vs Saline, ‡ P ˂ 0.05 vs 202 CaptoprilTM , ‡‡ P ˂ 0.01 vs CaptoprilTM 203 204 205
Figure 2b shows the reduction of DBP in SHR caused by the oral administration 206
of the whey fraction of milk fermented by specific L. lactis strains. The highest 207
decrement of DBP was observed at 6 h post oral administration. At the same time, no 208
significant difference was found (P ˂ 0.05) when SHR were treated with whey fraction 209
of milk fermented by L. lactis NRRL B-50571 at any protein content or whey fraction of 210
fermented milk L. lactis NRRL B-50572-5. Whey fractions from milk fermented by L. 211
lactis NRRL B-50571 as well as milk fermented with L. lactis NRRL B-50572 presented 212
an important dosage dependent antihypertensive effect through DBP measurements. 213
Although, CaptoprilTM
generated the maximum DBP reduction with each measurement, 214
there was not a significant difference (P ˂ 0.05) with the hypotensive effect of the whey 215
fraction of milk fermented by L. lactis NRRL B-50572-5. 216
L. lactis fermented milk hypotensive effect
70
217
218
219
220
L. lactis fermented milk hypotensive effect
71
Figure 2. Blood pressure and HR lowering effect in SHR treated with milk fermented by specific L. lactis 221
strains: (a) systolic blood pressure (SBP), (b) diastolic blood pressure (DBP) and (c) heart rate (HR). 222
Positive control CaptoprilTM
, negative control saline, whey fraction of milk fermented by L. 223
lactis NRRL B-50572-3 (35 mg protein/kg BW) , whey fraction of milk fermented by L. lactis NRRL 224
B-50571-3 (35 mg protein/kg BW) , whey fraction of milk fermented by L. lactis NRRL B-50572-5 225
(50 mg protein/kg BW) , whey fraction of milk fermented by L. lactis NRRL B-50571-5 (50 mg 226
protein/kg BW) . Data is shown by means with their standard error. Each SHR group had seven 227
animals. 228
229
HR reductions at 2, 4, 6 and 24 h of treated SHR are shown in figure 2c. There 230
was not a significant difference (P ˂ 0.05) in HR presented by rats administered with 231
whey fractions from milk fermented with L. lactis NRRL B-50572-5 or NRRL B-50571-232
3 or CaptoprilTM
. As in SBP and DBP, the lowest HR values were found at 6 h post 233
administration of treatments. In fact, SHR treated with the whey fraction L. lactis NRRL 234
B-50571-3 fermented milk, as well as the whey fraction L. lactis NRRL B-50572-5 235
fermented milk presented the maximal HR decrement, 16.6 ± 9.2 and 16.9 ± 11.5 beats 236
min-1
, respectively. Moreover, a significant (P ˂ 0.05) HR decrement (33.4 beats/min) 237
was found in SHR that received the whey fraction from L. lactis NRRL B-50572-5 238
fermented milk when compared with saline treatment at the end of the 24-h post oral 239
administration. 240
241
Tables 2 and 3 show the initial blood pressure and HR values, respectively. A 242
notorious difference on SBP values at 2 h (184.3 ± 2.8 mm Hg) and 24 h (203.9 ± 2.2 243
mm Hg) was found post administration on SHR treated with saline solution. These 244
results may be due to increasing hypertension of SHR at this age. A similar result was 245
found in HR measurements. SHR administered with saline presented 383.6 ± 22.1 246
beats/min 2 h after orally ingestion versus 412.0 ± 6.3 beats/min 24 h later. The 247
dispersion of the data may be due to the fact that SBP, DBP and HR measurements were 248
done on conscious SHR. 249
Table 3. Measurements of heart rate (HR) in spontaneously hypertensive rats (SHR) treated with milk fermented by specific
L. lactis fermented milk hypotensive effect
72
L. lactis strains at different times (Mean values with their standard error)
2 h 4 h 6 h 24 h
HR (beats/min)
HR (beats/min)
HR (beats/min) HR (beats/min)
Mean SEM Mean SEM Mean SEM Mean SEM
Saline 383.6 22.1
396.1 10.0
384.3 11.1
412.0 6.3
CaptoprilTM 396.2 13.6
375.7 10.4
376.7 6.0
389.5 5.9
NRRL B-50572-3 413.8 5.1
392.0 7.7
384.3 5.1
384.0 11.3
NRRL B-50572-5 379.6 11.9
371.3 11.6
371.3 11.5
378.4* 5.9
NRRL B-50571-3 382.2 8.2
375.9 7.0
371.6 9.2
386.8 16.2
NRRL B-50571-5 387.3 10.8 381.0 12.8 399.3 8.9 384.1 7.2
250 NRRL B-50572-3, milk fermented by L. lactis NRRL B-50572 (35 mg protein/kg BW); NRRL B-50572-5, milk fermented by L. 251 lactis NRRL B-50572 (50 mg protein/kg BW); NRRL B-50571-3, milk fermented by L. lactis NRRL B-50571 (35 mg protein/kg 252 BW); NRRL B-50571-5, milk fermented by L. lactis NRRL B-50571 (50 mg protein/kg BW). * P ˂ 0.05 vs Saline 253
254
Discussion 255
256
To date, SHR is one of the most widely utilized animal model to study essential 257
hypertension and associated metabolic disorders (Pavenec and Kurtz, 2010). Some of the 258
advantages of using SHR strains to evaluate primary hypertension are related to the 259
ability to provide new insights into relevant mechanisms for blood pressure control in 260
rodents and humans and to the fact that many genes identified in animal models have 261
been extensively studied in humans. Probably the most important issue of using SHR to 262
evaluate the antihypertensive effects of specific substances is the feasibility to determine 263
and characterize the influence of specific treatments establishing a close relationship 264
between antihypertensive substance-hypotensive effect (Saavedra 2009). In recent years, 265
peptides derived from food protein substrates such as milk, egg, fish, sesame, pea, sake, 266
rice and corn have demonstrated important antihypertensive activity (Hong et al., 2008). 267
268
At the beginning of this research we knew that specific L. lactis strains were able 269
to ferment milk with the ability to inhibit the activity of angiotensin I-converting 270
enzyme, which is associated to the reduction of blood pressure. In fact, these previous in 271
vitro studies showed the relevant capacity of L. lactis NRRL B-50571 and L. lactis 272
L. lactis fermented milk hypotensive effect
73
NRRL B-50572 to hydrolyze milk proteins to exert antihypertensive activity. On the 273
other side, based on the complexity of living organisms, a clear direct correlation 274
between the hypotensive activity in vitro and in vivo studies has not been reported. 275
Therefore, it was necessary to evaluate the antihypertensive effect milk fermented by 276
specific L. lactis strains on an animal model. 277
278
This study demonstrated the ability of specific L. lactis strains to ferment milk 279
with blood pressure and HR lowering effect in vivo. The remarkable hypotensive effect 280
as well as the HR reduction were observed at 6 h post oral administration. In fact, 281
Muguerza et al. (2006) reported maximal SBP and DBP reductions in SHR treated with 282
the whey fraction of milk fermented by Enterococcus faecalis 4 and 6 h post 283
administration. 284
285
An association of specific minerals with blood pressure reduction in SHR has 286
been reported (Whelton et al., 1997; Adachi et al., 1994). Therefore, it was necessary to 287
determine the chemical composition of the whey fractions from fermented milk (Table 288
1). Indeed, Civantos et al. (2004), reported a decrement in systolic and diastolic blood 289
pressure in SHR fed with an enriched-Ca diet (2.5%) during a long-term experiment. 290
However, in our experiment the dose with the highest Ca concentration corresponded to 291
less than 1% of the SHR diet. On the other side, the content of magnesium in the SHR 292
diet may play a role in hypertension. Sipola et al. (2001), did not report a blood pressure 293
lowering effect associated to magnesium content in SHR after a 12 week treatment. It is 294
important to notice that the amount of magnesium present in milk fermented by 295
Lactobacillus helveticus (0.33 mg/g) in that study was similar to the magnesium content 296
found in milk fermented by specific L. lactis in this study. Thus, the effect of Ca and Mg 297
content on blood pressure reduction of SHR of this study may not be important. 298
299
L. lactis fermented milk hypotensive effect
74
In this study, two protein concentrations were administered to SHR by a single 300
dose, 35 or 50 mg/kg BW. Hence, according to results shown in figure 2a, milk 301
fermented by L. lactis NRRL B-50572 was dose dependent. On the other hand, it is 302
important to notice that the maximal SBP reduction (17.7 ± 4 mm Hg) was observed in 303
SHR treated with the lowest dose of whey fraction from milk fermented by L lactis 304
NRRL B-50571, thus this treatment was not dose dependent. Nakamura et al (1995) 305
evaluated the capacity of milk fermented with Lactobacillus helveticus and Sacaromyces 306
cerevisiae to reduce SBP. SHR received 34 mg protein/kg BW of the whey fraction 307
resulting in a decrement of 21.8 ± 4.2 mm Hg. Masuda et al. (1996), also used the same 308
Lactobacillus helveticus and Sacaromyces cerevisiae strains to ferment milk by 309
increasing the protein content to 68 mg/kg BW and obtained – 26.4 ± 3.1 mm Hg. These 310
findings showed a dose dependent relationship. 311
312
On the other hand, Muguerza et al. (2006), also evaluated the antihypertensive 313
effect of milk fermented by Enterococcus(E.) faecalis in SHR. In this case, a single dose 314
of milk fermented by E. faecalis CECT 5727 and CECT 5728 presented the maximal 315
DBP reduction of 34.8 ± 4.5 mm Hg (Figure 2b). Chen et al. (2007) measured the 316
antihypertensive effect of fresh low-fat milk fermented by five mixed lactic acid 317
bacteria. The DBP value reported was - 21.5 mm Hg after 8 weeks of oral 318
administration. Similar results were found in SHR treated with milk fermented by L. 319
lactis NRRL B-50572-5 by a single dose (- 23.9 ± 9.4 mm Hg). 320
321
HR is a major determinant of myocardial oxygen consumption and cardiac work. 322
Indeed, high HR has been considered as an important risk factor for atherosclerosis, 323
therefore, its reduction may represent an important strategy for the treatment of patients 324
with a wide range of cardiac disorders (Palatini, 2009). In this study, an evident 325
reduction of the HR of SHR was observed after the single dose treatment (Figure 2c). 326
The whey fraction corresponding to milk fermented by L. lactis NRRL B-50571-3 327
L. lactis fermented milk hypotensive effect
75
decreased HR by 16.6 ± 9.2 beats/min, while the whey fraction of milk fermented by L. 328
lactis NRRL B-50572-5 decreased HR by 16.9 ± 11.5 beats/min at 6 h after oral 329
administration. On the other hand, CaptoprilTM
decreased HR by 11.4 ± 5.9 beats/min. 330
To the best of our knowledge this is the first report on the blood pressure and HR 331
lowering effect of whey fractions obtained from milk fermented by L. lactis. 332
333
In conclusion, the present study demonstrated the relevant blood pressure and 334
heart rate lowering effect in SHR of fermented milk with specific L. lactis strains. 335
Moreover, previous research in our laboratory reported the capacity of these strains to 336
biogenerate interesting aroma profiles. Thus, whey fractions from milk fermented by 337
specific L. lactis strains may be used as a functional ingredient or food with important 338
advantages in the prevention and treatment of cardiovascular disease associated to 339
hypertension. Ongoing research is being carried out to identify the antihypertensive 340
peptides sequences and their mechanism of absorption. 341
342
343
Acknowledgments 344
345
We would like to thank María del Carmen Estrada and Rodrigo Pacheco for their 346
technical support during the experiment. This study was supported by CONACYT 347
(projects 134295 and 42340-Z). The authors´ responsibilities were as follows: A.F. G-C. 348
looked for funding and provided technical expertise, H. A-G provided technical 349
expertise in the handling of animals and supported with the animal laboratory center, B. 350
V-C. looked for funding, revised the manuscript and provided technical expertise; J.C. 351
R-F. conducted the study, designed the experiment, analyzed data statistically and wrote 352
the manuscript. 353
All authors declare that there is not conflict of interests. 354
L. lactis fermented milk hypotensive effect
76
References 355
356
Adachi M, Nara Y, Mano M, et al. (1994) Effect of dietary magnesium supplementation 357
on intralymphocytic free calcium and magnesium in stroke-prone 358
spontaneously hypertensive rats. Clin Exp Hypertens 16, 317-326. 359
Aihara K, Kajimoto O, Hirata H, et al. (2005) Effect of powdered fermented milk with 360
Lactobacillus helveticus on subjects with high-normal blood pressure or mild 361
hypertension. J Am Coll Nutr 24, 257-265. 362
Anadón A, Martínez MA, Ares I, et al. (2010) Acute and repeated dose (4 weeks) oral 363
toxicity studies of two antihypertensive peptides, RYLGY and AYFYPEL, that 364
correspond to fragments (90–94) and (143–149) from αs1-casein. Food & Chem 365
Toxicol 48, 1836-1845. 366
Ayad Eman HE (2009) Starter culture development for improving safety and quality of 367
Domiati cheese. Food Microbiol 26, 533-541. 368
Chen GW, Tsai JS & Pan SB (2007) Purification of angiotensin I-converting enzyme 369
inhibitory peptides and antihypertensive effect of milk produced by protease-370
facilitated lactic fermentation. Int Dairy J 17: 641-647. 371
Civantos B & Aleixandre A (2004) Blood pressure and α-vascular reactivity in 372
hypertensive rats treated with amlodipine and dietary Ca. Eur J Pharmacol 489, 373
101-110. 374
Gutiérrez-Méndez N, Vallejo-Cordoba B, González-Córdova AF, et al. (2008) 375
Evaluation of aroma generation of Lactococcus lactis with an electronic nose 376
and sensory analysis. J Dairy Sci 91,49-57. 377
Hong F, Ming L, Yi S, et al. (2008) The antihypertensive effect of peptides: A novel 378
alternative to drugs? Peptides 29, 1062-1071. 379
Hugenholtz J (2008). The lactic acid bacterium as a cell factory for food ingredient 380
production. Int Dairy J 18, 466-475. 381
L. lactis fermented milk hypotensive effect
77
Jauhiainen T, Wuolle K, Vapaatalo H, et al. (2007) Oral absorption, tissue distribution 382
and excretion of a radiolabelled analog of a milk-derived antihypertensive 383
peptide, Ile-Pro-Pro, in rats. Int Dairy J 17, 1216-1223. 384
385
Korhonen H (2009) Milk-derived bioactive peptides: From science to applications. J 386
Funct Foods 1, 177- 187. 387
Leclerc PL, Guthier SF, Bachelard H, et al. (2002) Antihypertensive activity of casein-388
enriched milk fermented by Lactobacillus helveticus. Int Dairy J 12, 995-1004. 389
López-Fandiño R, Otte J & van Camp J (2006) Physiological, chemical technological 390
aspects of milk-protein-derived peptides with antihypertensive and ACE-391
inhibitory activity. Int Dairy J 16, 1277-1293. 392
Chobanian A, et al. (2003). The National High Blood Pressure Education Program 393
Coordinating Committee, 7th
Report of the Joint National Committee on 394
Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. J 395
Am Med Assoc 289, 2560-2572. 396
Muguerza B, Ramos M, Sánchez E, et al. (2006) Antihypertensive activity of milk 397
fermented byEnterococcus faecalis strains isolated from raw milk. Int Dairy J 398
16, 61-69. 399
Nakamura Y, Yamamoto N, Sakai K et al. (1995) Antihypertensive effect of sour milk 400
and peptides isolated from it that are inhibitors to angiotensinI-converting 401
enzyme. J Dairy Sci 78, 1253-1257. 402
Tanase H, Yamori Y, Hansen CT et al. (1982) Heart size in inbread strains of rats. Part 403
1. Genetic determination of the development of cardiovascular enlargement in 404
rats. Hypertension 4, 864-872. 405
L. lactis fermented milk hypotensive effect
78
Odamaki T, Yonezawa S, Sugahara H et al. (2011) A one step genotypic identification 406
of Lactococcus lactis subspecies at the species/strain levels. Systematic Appl 407
Microbiol 34, 429-434. 408
Okamoto K & Aoki K (1963) Development of a strain of spontaneously hypertensive 409
rats. Jpn Circ Soc 27, 282-293. 410
Palatini P (2009) Elevated heart rate in cardiovascular diseases: A target for treatment?. 411
Prog Cardio-vasc Dis 52, 46-60. 412
Pravenec M & Kurtz T (2010) Recent advances in genetics of Spontaneously 413
Hypertensive Rat. Curr Hypertens Rep 12, 5-9. 414
Quirós A, Ramos M, Muguerza B, et al (2007). Identification of novel antihypertensive 415
peptides in milk fermented with Enterococcus faecalis. Int. Dairy J. 17, 33-416
41. 417
Safar ME, Levy BL & Struijker-Boudier HAJ (2003) Current perspectives on arterial 418
stiffness and pulse pressure in hypertension and cardiovascular diseases. 419
Circulation 107, 2864-2869. 420
Skeggs LT, Kahn JR & Shumway NP (1956) The preparation and function of the 421
hypertension-converting enzyme. J Exp Med 103, 295-299. 422
Stanley SF (2007) The importance of diastolic blood pressure in predicting 423
cardiovascular risk. J Am Soc Hypertens 1, 82-93. 424
Saavedra J (2009) Opportunities and limitations of genetic analysis of hypertensive rat 425
strains. J Hypertens 27, 1129-1133. 426
Whelton P, He J., Cutler J, et al (1997) Effects of oral potassium on blood pressure. 427
Meta-analysis of randomized controlled clinical trials. J Am Med Assoc 277, 428
1624-1632. 429
Yamamoto N, Maeno M & Takano T (1999) Purification and charaterization of an 430
antihypertensive peptide from a yogurt-like product fermented. J Dairy Sci 82, 431
1388-1393.432
Capítulo 5
Antihypertensive and hypolipidemic effect
of milk fermented by specific
Lactoccocus lactis strains
Artículo por enviarse al: Journal of Functional Foods
Antihypertensive and hypolipidemic L. lactis fermented milk
80
Antihypertensive and hypolipidemic effect of milk fermented 1
by specific Lactococcus lactis strains 2
3
J.C. Rodríguez-Figueroa, A.F. González-Córdova, H. Astiazaran-García, 4
B. Vallejo-Cordoba* 5
6
Centro de Investigación en Alimentación y Desarrollo A.C. (CIAD, A.C.). 7
Carretera a la Victoria Km. 0.6, 83304, Hermosillo, México 8
* Corresponding Author. 9
Belinda Vallejo-Cordoba 10
Centro de Investigación en Alimentación y Desarrollo A.C. (CIAD, A.C.). 11
Carretera a la Victoria Km. 0.6, 83304, Hermosillo, México 12
Phone: +52 (662) 289- 24-00 ext. 303; Fax: +52 (662) 280- 04-21. 13
E-mail address: [email protected] 14
15
16
17
18
Antihypertensive and hypolipidemic L. lactis fermented milk
81
ABSTRACT 19
The antihypertensive and hypolipidemic effects of milk fermented by specific 20
Lactococcus (L.) lactis strains in spontaneously hypertensive rats (SHR) 21
were investigated. SHR were feed ad libitum with milk fermented by L. lactis 22
NRRL B-50571, L. lactis NRRL B-50572, CaptoprilTM (40 mg/ kg body 23
weight) or purified water. Results suggested that L. lactis fermented milks 24
presented blood pressure-lowering effect. There was not a significant 25
difference (p > 0.05) among milk fermented by L. lactis NRRL B-50571 and 26
CaptoprilTM by the second and third week of treatment. Additionally, milk 27
fermented by L. lactis strains modified SHR lipid profiles. Milk fermented by 28
L. lactis NRRL B-50571 and B-50572 were able to reduce plasma low-29
density lipoprotein (LDL) cholesterol by 55.4 ± 3 mg/dL and 66.3 ± 4 mg/dL, 30
respectively. In conclusion, milk fermented by L. lactis strains may be a 31
coadyuvant in the reduction of hypertension and hyperlipidemia. 32
33
Key words 34
Lactococcus lactis, fermented milk, blood pressure, antihypertensive and 35
hypolipidemic effect 36
37
38
39
Antihypertensive and hypolipidemic L. lactis fermented milk
82
1. Introduction 40
Coronary heart disease (CHD), which is considered the most common and 41
serious form of cardiovascular disease, is the first cause of death in 42
development industrialized countries (Chobanian et al., 2003). Hypertension 43
and elevated blood cholesterol levels, particularly high low density-density 44
lipoprotein cholesterol (LDL-C), are two of the major modified risk factors to 45
develop CHD (Department of Health and Human Services, 2000). 46
The use of animal models to study the effect of food derived substances on 47
CHD has been quite accepted nowadays. Spontaneously hypertensive rat 48
(SHR) strain is characterized by presenting hyperlipidemia, hypertension, 49
hyperinsulinemia and diabetes type 2 (Brown et al., 2011). These 50
characteristics, besides the similarity of the pathologies mechanism in 51
humans, made SHR one of the most utilizable models (Doggrell & Brown, 52
1998). Therefore, several studies have focused on the evaluation of 53
antihypertensive and hypolipidemic effects of functional foods in SHR (Manso 54
et al., 2008;Turpeinen et al., 2009; Pal et al., 2010) . 55
Milk proteins have received increased attention as potential ingredients in 56
health-promoting functional foods. It is accepted that proteins from milk may 57
act as precursors of biologically active peptides with different physiological 58
effects on the digestive, endocrine, cardiovascular, immune and nervous 59
systems (Korhonen 2009). Indeed, it has been reported that an effective way 60
to increase the amount of bioactive peptides in dairy products is by milk 61
Antihypertensive and hypolipidemic L. lactis fermented milk
83
fermentation with highly proteolytic strains of lactic acid bacteria (LAB) 62
(López-Fandiño et al., 2006). LAB growth in milk is dependent on the specific 63
proteolytic and peptidolytic systems for the generation of free amino acids 64
and free peptides as a source of nitrogen (Hugenholtz, 2008). In fact, several 65
studies suggested that peptides released by Enterococcus faecalis strains 66
from milk proteins were able to decrease arterial blood pressure in 67
spontaneously hypertensive rats (SHR) (Muguerza et al., 2006; Quirós et al., 68
2007). 69
L. lactis is one of the most well studied LAB because of its importance 70
as part of commercial starter cultures used in the manufacture of fermented 71
dairy products (Odamaki et al., 2011). It has been reported that L. lactis 72
strains are able to improve the organoleptic characteristics of dairy products 73
(Ayad 2009). Previous studies showed that specific L. lactis strains isolated 74
from native ecosystems were able to produce remarkable aroma profiles in 75
fermented milk (Gutiérrez-Méndez et al., 2008). In addition, fermented milks 76
with these specific strains were able to inhibit ACE activity. However, the 77
antihypertensive and hypolipidemic effects of fermented milk with specific L. 78
lactis strains has not been tested in vivo. Therefore, the objective of this 79
research was to evaluate the antihypertensive and hypolipidemic effects of 80
milk fermented by specific L. lactis strains through a long-term study in SHR. 81
82
83
Antihypertensive and hypolipidemic L. lactis fermented milk
84
2. Materials and methods 84
2.1. L. lactis strains 85
Two L. lactis strains, NRRL B-50571 and NRRL B-50572, obtained from the 86
Dairy Laboratory collection at Centro de Investigación en Alimentación y 87
Desarrollo, A.C (CIAD, Hermosillo, Sonora, Mexico) were deposited at the 88
Agricultural Research Service Culture Collection (NRRL) from the U.S. 89
Department of Agriculture. The strains were propagated in 10 mL of sterile 90
lactose (5 g L-1) M17 broth (DIFCO Sparks, MD, USA) and incubated at 30°C 91
for 24 h. Fresh cultures were obtained by repeating the same procedure. 92
Initial starter cultures were prepared by allowing L. lactis strains to reach 93
106-107 colony-forming units (cfu) mL-1 as enumerated on M17 agar 94
containing lactose (5 g L-1) (DIFCO Sparks, MD, USA). 95
96
2.2. Sample preparation 97
Organic (U.S. Department of Agriculture) grade A nonfat dry milk from 98
Organic Valley® (La Farge, WI, USA) was reconstituted (10 %, w/w) and 99
sterilized (100°C, 20 min). Every L. lactis strain was inoculated (7-8 log of 100
cfu/ml) in sterilized milk to obtain pre-cultures. The inoculated milk was 101
incubated for 12 h at 30°C. Pre-cultures were added (3% v/v) to sterilized 102
milk to get the different fermented milk batches. Incubation was performed at 103
30°C during 48 h. The fermentation process was stopped by heating at 98°C 104
Antihypertensive and hypolipidemic L. lactis fermented milk
85
for 10 min to inactive proteases and L. lactis strains (Guan-Wen et al., 2007). 105
Subsequently, samples were frozen at - 20°C. All fermented milk samples 106
were daily unfrozen and homogenized (Model 4169, Braun, Spain) for 20 107
minutes before use. Total protein (Method 960.52 AOAC, 1998), calcium, 108
magnesium and potassium (EPA 3052), total fat (Method 942.05 AOAC, 109
1998) and lactose (Method 930.28 AOAC, 1998) contents were evaluated 110
(Table 1). 111
112
2.3. In vivo experimental protocol 113
114
Thirty-two male SHR were obtained from Harlan Laboratories, INC, 115
(Indianapolis, IL, USA). The rats were randomly housed in pairs per cage at 116
21 ± 2°C with 12 h light/dark cycles, 52 ± 6 % relative humidity and with ad 117
libitum intake of a standard diet (Teklad, Harlan Laboratories, USA) during 118
the experiment. SHR (27-28 weeks old and weighting 355 ± 24 g) were 119
divided into four groups of eight rats (n = 8): purified water was used as the 120
negative control, CaptoprilTM (proven hypotensive drug) (40 mg/kg body 121
weight (BW) was the positive control, milk fermented by L. lactis NRRL B-122
50571 and milk fermented by L. lactis NRRL 50572. All SHR had free access 123
to each treatment during three weeks as part of the protocol. Half of the 124
animals were sacrificed at the end of that period. The rest of the SHR only 125
received purified water during one more week before being sacrificed. A 126
Antihypertensive and hypolipidemic L. lactis fermented milk
86
research animal protocol was followed according to the guidelines 127
established by the institutional Ethics Committee. 128
129
130
2.4. Antihypertensive effect measurements 131
132
The lowering blood pressure effect of milk fermented by specific L. lactis 133
strains on SHR was monitored through time. Animals were deposited in 134
restrainers in the warming chamber for 20 min at 32°C to detect pulsations 135
through the caudal artery. Systolic (SBP) and diastolic (DBP) blood 136
pressures were measured five times on each conscious animal before 137
treatments and every week during the experiment. Measurements were 138
obtained using the tail-cuff method between 9 and 12 h to eliminate circadian 139
cycles. The non-invasive blood pressure system used in this experiment 140
included a photoelectric sensor, an amplifier, an automatic inflation cuff and 141
software (Model 229, IITC, USA). 142
143
144
2.5. Plasma lipid profile evaluation 145
146
The hypolipidemic activity of milk fermented by specific L. lactis strains were 147
also evaluated in SHR. Blood samples were collected under anesthesia by 148
cardiac puncture in tubes with heparin (Sarstedt, Germany). Subsequently, 149
Antihypertensive and hypolipidemic L. lactis fermented milk
87
samples were centrifugated at 2,500 rpm, 4°C for 10 min to obtain the 150
plasma and they were frozen at -20°C for further studies. Tryglicerides (TG), 151
total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) levels 152
in plasma were determined by the Randox Labs commercial kit (UK), while 153
low density lipoprotein cholesterol (LDL-C) was calculated as the difference 154
between TC and HDL-C according to specifications. 155
156
157
2.6. Statistical analysis 158
Normality of experimental data was evaluated as a prerequisit for the 159
analysis by one- way ANOVA. Differences between means were assesed by 160
the Tuckey-Kramer multiple-comparison test and they were considered 161
significantly different when P < 0.05. Results were processed by the NCSS 162
2007 statistical program. Data were presented as means values ± standard 163
errors (S.E.M). 164
165
3. Results and discussion 166
167
3.1. Nutritional composition of L. lactis fermented milk 168
169
Nutritional composition corresponding to fermented milks are presented in 170
table 1. Samples were not significantly different between them (p > 0.05). 171
Antihypertensive and hypolipidemic L. lactis fermented milk
88
One of the most important macronutrient in fermented milk, which is related 172
to the antihypertensive activity, may be the proteins. They eventually could 173
be hydrolyzed by the proteolytic and peptidolytic systems of L. lactis strains 174
to form hypotensive peptides (López-Fandiño et al., 2006). However, results 175
showed that protein content in both fermented milks were similar. On the 176
other side, the presence of calcium in dairy products has been associated to 177
an antihypertensive effect (Jäkäla et al., 2009). The content of calcium in milk 178
fermented by L. lactis NRRL B-50571 and L. lactis NRRL B-50571 B-50572 179
were 368 ± 11.3 and 366 ± 3.0 mg /100 g, respectively. According to Sipola 180
et al. (2002) the antihypertensive effect of fermented milk products was 181
attributed to a factor other than calcium. It has been reported that diets 182
including more than 2.5% of calcium reduce arterial blood pressure (Civantos 183
& Aleixandre, 2007). In this study, the content of calcium in the diet was 0.81-184
0.83 %, thus the antihypertensive effect may not attributed to calcium. 185
186
3.2. Blood pressure-lowering effect 187
188
Before the experiment, SHR (26-27 weeks old) systolic and diastolic blood 189
pressures were 226 ± 3.2 and 180 ± 4.5 mm Hg, respectively. Both L. lactis 190
fermented milks were able to reduce blood pressure during the experiment 191
(figure 1 and 2). Results did not show significant difference (p > 0.05) 192
between systolic blood pressure (SBP) masurements in the first week (figure 193
1). However, by the second week, the SBP reduction in SHR that received 194
Antihypertensive and hypolipidemic L. lactis fermented milk
89
milk fermented by L. lactis NRRL B-50571 (-20.2 ± 3.8 mm Hg) was not 195
statistically different (p > 0.05) from those that received CaptoprilTM (-30.1 ± 196
7.1 mm Hg). In fact, by the second and third week, SHR treated with milks 197
fermented by L. lactis NRRL B-50571 or B-50572 or CaptoprilTM presented a 198
marked lowering-effect on SBP. By the fourth week of treatment, milk 199
fermented by L. lactis NRRL B-50571 was able to reduce SBP by 23.3 ± 1.8 200
mm Hg, meanwhile CaptoprilTM reduce SBP by 28.1 ± 1.8 mm Hg. These 201
results were similar to those reported by others, who found that milk 202
fermented by Lactobacillus helveticus LBK16H was able to reduce SBP by 203
21 mm Hg (Sipola et al., 2002). As it is observed in figure 1, the SBP 204
lowering-effect in SHR treated with milk fermented by L. lactis NRRL B-205
50571 increases with time. Indeed, the maximal SBP reduction was found by 206
the fourth week, even though animals drank only water in the last week. 207
Therefore, these results suggest a residual SBP lowering-effect after 208
cessation of the treatment. Jauhiainen et al. (2007) demonstrated the 209
presence of a radiolabelled analog of milk-derived antihypertensive peptide, 210
Ile-Pro-Pro, in several rat tissues 48 h after a single oral administration. 211
Therefore, milk fermented containing antihypertensive peptides administered 212
for longer treatments may extend their bioactivity for longer periods. 213
214
Diastolic blood pressure (DBP) was also monitored as part of the 215
experimental procedure. SHR treated with milk fermented by L. lactis NRRL 216
B-50571 and B-50572 presented DBP lowering-effect during the experiment 217
Antihypertensive and hypolipidemic L. lactis fermented milk
90
(figure 2). As in SBP, the first week DBP measurements were not 218
significantly different (p > 0.05) between treatments. By the second week, 219
milk fermented by L. lactis NRRL B-50571 was able to reduce DBP by 24.5 ± 220
6.6 mm Hg. Meanwhile, CaptoprilTM reduced DBP by 38.4 ± 8.5 mm Hg. By 221
the third experimental week, the DBP lowering-effect was not significantly 222
different (p > 0.05) between SHR treated with CaptoprilTM and milk fermented 223
by L. lactis NRRL B-50571 or L. lactis NRRL B-50572. The most important 224
DBP reduction (49.8 ± 3.5 mm Hg) was observed by the fourth week of 225
treatment in SHR that received milk fermented by L. lactis NRRL B-50571. 226
227
228
3.3. Lipidic profile 229
230
Results suggested that fermented milks were able to modified remarkably 231
SHR lipid profiles by the third week of treatment. SHR that received milks 232
fermented by L. lactis NRRL B-50571 and B-50572 presented 55.4 ± 3 mg/dL 233
and 66.2 ± 4 mg/dL reduction of low-density lipoprotein cholesterol (LDL-C), 234
respectively, when compared to SHR administered with purified water (figure 235
3). Similarly, results showed that milk fermented by L. lactis strains reduced 236
HDL-C significantly (p ˂ 0.05) in SHR treated (figure 4). According to the 237
National Cholesterol Education Program (NCEP) Adult Treatment Panel III, it 238
is a priority to reduce LDL-C as a primary target to reduce the risk of heart 239
disease and as a secondary target to reduce the risk of metabolic syndrome 240
Antihypertensive and hypolipidemic L. lactis fermented milk
91
(Expert Panel on Detection, Evaluation, and Treatment of High Blood 241
Cholesterol in Adults, 2001). The lowering effect on LDL-C observed in this 242
study may be attributed to the ingestion by SHR of dairy protein including 243
whey protein. It has been reported that obese and overweight individuals who 244
consume whey protein for 12 weeks presented an important reduction in 245
LDL-C (Pal et al. 2010). 246
247
Plasma triglyceride (TG) content was also decreased by 34.7 ± 3.7 mg/dL in 248
SHR treated with L. lactis NRRL B-50572 fermented milk when compared to 249
purified water (figure 5). Additionally, plasma total cholesterol (TC) content 250
was also reduced in SHR treated animals. Milks fermented by L. lactis NRRL 251
B-50572 and L. lactis NRRL B-50571 were able to reduce TC by 10 ± 3.2 252
mg/dL and 8.6 ± 2.4 mg/dL, respectively (figure 6). It has been reported that 253
milk supplemented with whey protein concentrate and fermented by 254
Lactobacillus casei TMC0409 and Streptococcus thermophilus TMC1543 255
decreased serum TG from 151.7 ± 13.9 mg/dL to 115.7 ± 11.1 mg/dL in 256
humans, after 4 weeks of treatment (Kawase et al., 2000). 257
258
Even though, SHR lipid profile was also evaluated after one week of being 259
treated with purified water, there was not a significant difference among 260
treatments (p > 0.05). Thus, these results suggest that there was not a 261
residual hypolipidemic lowering-effect after cessation of the treatment. 262
263
Antihypertensive and hypolipidemic L. lactis fermented milk
92
4. CONCLUSION 264
265
Milk fermented by specific L. lactis strains were able to reduce systolic and 266
diastolic blood pressure in a SHR strain. Moreover, SHR lipid profile was 267
improved by fermented milk intake for three weeks. Thus, the use of milk 268
fermented by specific lactic acid bacteria may be considered as a coadyuvant 269
for the improvement of cardiovascular health. 270
271
Acknowledgements 272
273
We would like to thank María del Carmen Estrada, Bertha I. Pacheco 274
Moreno, Ana Cristina Gallegos and Rodrigo Pacheco for their technical 275
support during the experiment. This study was supported by the Mexican 276
Council of Science and Technology Research CONACYT (projects 42340-Z 277
and 134295). 278
279
280
281
Antihypertensive and hypolipidemic L. lactis fermented milk
93
REFERENCES 282
283
Ayad, E.H.E. (2009). Starter culture development for improving safety and 284
quality of Domiati cheese. Food Microbiology, 26, 533-541. 285
Brown L.M., Hansen C.T., & Huberty A.F. (2011). Traits of the metabolic 286
syndrome alter corpulent obesity in LAN, SHR and DSS rats: 287
Behavioral and metabolic interactions with adrenalectomy. Physiology 288
& Behavior, 103, 98-103. 289
Chobanian, A., Bakris, G., Black, H., Cushman, W., Green, L., Izzo, J., 290
Jones, D., Materson, B., Oparil, S., Wright, J., Roccella, E., & the 291
National High Blood Pressure Education Program Coordinating 292
Committel. (2003). The National High Blood Pressure Education 293
Program Coordinating Committee, 7th Report of the Joint National 294
Committee on Prevention, Detection, Evaluation, and Treatment of 295
High Blood Pressure. Journal of American Medical Association, 289, 296
2560-2572. 297
Civantos, B., & Aleixandre, A. (2004). Blood pressure and α-vascular 298
reactivity in hypertensive rats treated with amlodipine and dietary Ca. 299
European Journal of Pharmacology, 489, 101-110. 300
Department of Health and Human Services, Public Health Service, Healthy 301
People 2010, Vol. I, U.S. Government Printing Office, Washington, 302
D.C., December, 2000. 303
Antihypertensive and hypolipidemic L. lactis fermented milk
94
Doggrell, S., & Brown, L. (1998). Rat models of hypertension, cardiac 304
hypertrophy and failure. Cardiovascular Research, 39, 89-105. 305
Expert Panel on Detection, Evaluation, and Treatment of High Blood 306
Cholesterol in Adults, Executive Summary of the Third Report of the 307
National Cholesterol Education Program (NCEP) (Adult Treatment 308
Panel III). (2001). Journal of the American Medical Association, 285, 309
2486-2497. 310
Gutiérrez-Méndez, N., Vallejo-Cordoba, B., González-Córdova, A.F., 311
Nevárez-Moorillón, G.V., & Rivera-Chavira, B. (2008). Evaluation of 312
aroma generation of Lactococcus lactis with an electronic nose and 313
sensory analysis. Journal of Dairy Science, 91,49-57. 314
Guan-Wen, C., Jenn-Shou, T., & Bonnie Sun, P. (2007). Purification of 315
angiotensin I-converting enzyme inhibitory peptides and 316
antihypertensive effect of milk produced by protease-facilitated lactic 317
fermentation. International Dairy Journal, 17, 641-647. 318
Hugenholtz, J. (2008). The lactic acid bacterium as a cell factory for food 319
ingredient production. International Dairy Journal, 18, 466-475. 320
Jauhiainen, T., Wuolle, K, Vapaatalo, H., Kerojoki, O., Nurmela, K., Lowrie, 321
C., & Korpela, R. (2007). Oral absorption, tissue distribution and 322
excretion of a radiolabelled analog of a milk-derived antihypertensive 323
peptide, Ile-Pro-Pro, in rats. International Dairy Journal, 17 ,1216-324
1223. 325
Antihypertensive and hypolipidemic L. lactis fermented milk
95
Jäkälä, P., Jauhiainen, T., Korpela, R., & Vapaatalo, H. (2009). Milk protein-326
derived bioactive tripeptides Ile-Pro-Pro and Val-Pro-Pro protect 327
endothelial function in vitro in hypertensive rats. Journal of Functional 328
Foods, 1, 266-273. 329
Kawase, M., Hashimoto, H., Hosoda, M., Morita, H., & Hosono, A. (2000). 330
Effect of administration of fermented milk containing whey protein 331
concentrate to rats and healthy men on serum lipids and blood 332
pressure. Journal of Dairy Science, 83, 255-263. 333
Korhonen, H. (2009). Milk-derived bioactive peptides: From science to 334
applications. Journal of Functional Foods, 1, 177- 187. 335
López-Fandiño, R., Otte, J., & van Camp, J. (2006). Physiological, chemical 336
technological aspects of milk-protein-derived peptides with 337
antihypertensive and ACE-inhibitory activity. International Dairy 338
Journal, 16, 1277-1293. 339
Manso, M., Miguel, M., Even, J., Hernández, R., Aleixandre, A., & López-340
Fandiño, R. (2008). Effect of the long-term intake of an egg white 341
hydrolysate on the oxidative status and blood lipid profile of 342
spontaneously hypertensive rats. Food Chemistry, 109, 361-367. 343
Muguerza, B., Ramos, M., Sánchez, E., Manso, M.A., Miguel, M., Aleixandre, 344
A., Delgado, M.A., & Recio, I. (2006). Antihypertensive activity of milk 345
fermented by Enterococcus faecalis strains isolated from raw milk. 346
International Dairy Journal, 16:61-69. 347
Antihypertensive and hypolipidemic L. lactis fermented milk
96
Odamaki, T., Yonezawa, S., & Sugahara, H. (2011). A one step genotypic 348
identification of Lactococcus lactis subspecies at the species/strain 349
levels. Systematic Applied Microbiology, 34, 429-434. 350
Pal, S., Ellis, V., & Dhaliwal, S. (2010). Effects of whey protein isolate on 351
body composition, lipids, insulin and glucose in overweight and obese 352
individuals. British Journal of Nutrition, 104, 716-723. 353
Quirós, A., Ramos, M., Muguerza, B., Delgado, M., Miguel, M., Aleixandre, 354
A., & Recio, I. (2007). Identification of novel antihypertensive peptides 355
in milk fermented with Enterococcus faecalis. International Dairy 356
Journal, 17, 33-41. 357
Sipola, M., Finckenberg, P., Korpela, R., Vapaatalo, H., & Nurminen, M.-L. 358
(2002). Effects of long-term intake of milk products on blood pressure 359
in hypertensive rats. Journal of Dairy Research, 69, 103-111. 360
Turpeinen, A.M., Kampu, M., Rönnback, M., Seppo, L., Kautiainen, H., 361
Jauhiainen, T., Vapaatalo, H., & Korpela, R. (2009). Antihypertensive 362
and cholesterol-lowering effects of a spread containing bioactive 363
peptides IPP and VPP and plant sterols. Journal of Functional Foods, 364
1, 260-265. 365
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Table 1- L. lactis NRRL fermented milks nutritional composition. 366
B-50571 B-50572 Food1
Energy (kcal/100 g) 62.4 ± 0.5 65.6 ± 0.3 307
Protein (g/100 g) 3.1 ± 0.2 3.3 ± 0.1 18.6
Fat (g/100 g) ND ND 6.2
Carbohydrate (g/100 g) 12.5 ± 0.3 13.1 ± 0.2 44.2
Calcium (mg/100 g) 368 ± 11.3 366 ± 3.0 1000
Potasium (mg/100 g) 439 ± 14.6 434 ± 5.2 600
Magnesium (mg/100 g) 32.2 ± 1.2 31.1 ± 0.3 200
1 Data according Teklad Global 18% protein rodent diet, Harlan Laboratories (USA) ND = Not detected 367
368
Figure captions 369
370
Figure 1. SHR systolic blood pressure treated by L. lactis fermented milk. L. 371
lactis NRRL B-50571 fermented milk ; L. lactis NRRL B-50572 372
fermented milk ; CaptoprilTM = Positive control ; Purified water = 373
Negative control . Data is shown by mean values ± SEM ( n = 8). FM = 374
Fermented milk 375
376
Figure 2. SHR diastolic blood pressure treated by L. lactis fermented milk. L. 377
lactis NRRL B-50571 fermented milk ; L. lactis NRRL B-50572 378
fermented milk ; CaptoprilTM = Positive control ; Purified water = 379
Negative control . Data is shown by mean values ± SEM ( n = 8). FM = 380
Fermented milk 381
382
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Figure 3. Plasma low-density lipoprotein cholesterol in SHR treated by L. 383
lactis fermented milk. CaptoprilTM = Positive control; Purified water = 384
Negative control. Data is shown by mean values ± SEM (n = 8). 385
Figure 4. Plasma high-density lipoprotein cholesterol in SHR treated by L. 386
lactis fermented milk. CaptoprilTM = Positive control; Purified water = 387
Negative control. Data is shown by mean values ± SEM (n = 8). 388
Figure 5. Plasma triglycerides in SHR treated by L. lactis fermented milk. 389
CaptoprilTM = Positive control; Purified water = Negative control. Data is 390
shown by mean values ± SEM (n = 8). 391
Figure 6. Plasma total cholesterol in SHR treated by L. lactis fermented milk. 392
CaptoprilTM = Positive control; Purified water = Negative control. Data is 393
shown by mean values ± SEM (n = 8). 394
395
Figure 1 396
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Figure 2 399
Antihypertensive and hypolipidemic L. lactis fermented milk
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Figure 3 403
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Figure 4 406
Antihypertensive and hypolipidemic L. lactis fermented milk
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Figure 5 408
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Figure 6 410
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