centro de investigación en alimentación y desarrollo, a.c. · miocardio, enfermedades vasculares...

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.

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

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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

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

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

mailto:[email protected]


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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

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Purification and characterization of angiotensin I-converting enzyme inhibitors 347

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antihypertensive peptide from a yogurt-like product fermented by Lactobacillus 376

helveticus CPN4. J. Dairy Sci., 82:1388-1393. 377

378

379

380


53

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


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


55

Figure 1 405

406

Minutes 407

408

Figure 2 409

410


56

411

Figure 3 412

413

414

Figure 4 415

416


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

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


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



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


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


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


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


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


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


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


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


70

217

218

219

220


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


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


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


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


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


76

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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


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



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


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


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


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


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


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


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


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


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


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


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


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


93

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283

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Hugenholtz, J. (2008). The lactic acid bacterium as a cell factory for food 319

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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


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Jäkälä, P., Jauhiainen, T., Korpela, R., & Vapaatalo, H. (2009). Milk protein-326

derived bioactive tripeptides Ile-Pro-Pro and Val-Pro-Pro protect 327

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Kawase, M., Hashimoto, H., Hosoda, M., Morita, H., & Hosono, A. (2000). 330

Effect of administration of fermented milk containing whey protein 331

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López-Fandiño, R., Otte, J., & van Camp, J. (2006). Physiological, chemical 336

technological aspects of milk-protein-derived peptides with 337

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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

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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


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Odamaki, T., Yonezawa, S., & Sugahara, H. (2011). A one step genotypic 348

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levels. Systematic Applied Microbiology, 34, 429-434. 350

Pal, S., Ellis, V., & Dhaliwal, S. (2010). Effects of whey protein isolate on 351

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1, 260-265. 365


97

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


98

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

397

398

Figure 2 399


99

400

401

402

Figure 3 403

404

405

Figure 4 406


100

407

Figure 5 408

409

Figure 6 410

411

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