anti hypertensive pro bio tics

8/14/2019 Anti Hypertensive Pro Bio Tics

1/31

Antihypertensive activity of milkfermented byEnterococcus faecalis strains

isolated from raw milk

RAJESH KUMARRAJESH KUMARPh.D 1Ph.D 1stst yryrDairy Microbiology DivisionDairy Microbiology Division

N.D.R.IN.D.R.I


2/31

Nov 15,2005.

Introduction

Hypertension :- Cardiovascular diseases.

Renin-angiotensin-aldosterone system :- Maintains arterial blood pressure. Angiotensin-converting enzyme is main component.

(Ondetti et al, 1977) Angiotensin I(inactive decapeptide)

ACE Angiotensin II

(active octapeptide)

Active Bradykinin inactive Bradykinin

Vasodilation

X


3/31

Cont.

Inhibition of ACE generate Antihypertensiveeffect.

ACEI-drugs are commonly used.

Milk fermentation, shown to be a successful

strategy to produce ACEI peptides. Korhonen et al, 2003; Gobbetti et al 2004; Silva & Malcata, 2005

Lactibacillushelveticus Lb. delbrueckiisubsp. bulgaricus.

Milk fermented with Lb. helveticusLBK-16hhasdemonstrated significant antihypertensive effectsin humans.

Seppo et al, 2002; Seppo et al, 2003.


4/31

Cont..

Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP):-ACEI

peptides.

Source of IPP & VPP :- casein hydrolysates.

Other peptides of ACEI activity also isolated andcharacterized.


5/31

AIM

To select wild strains of bacteria from raw cowsmilk which were able to produce fermented milk withinhibitory ACE activity.

Antihypertensive effect of fermented milkproduced with four selected strains of E. faecalison

the arterial blood pressure of SHR and normotensiveWistar-Kyoto rats (WKY).


6/31

Materials and Methods

Substrates & Chemicals:-

I.)Hippuryl-L-histidyl-L-leucine& obtained from Sigma Chemical Co

Rabbit lung powder containing ACE.II.)FDA-Grade A skim milk.

III.)Distilled water from Millipore Milli-Q system.

Screening of microorganisms:-

Odor characteristics of raw milk samples(42 C for 48 h).

Initially incubated raw milk samples were chosen for microorganismisolation.


7/31

Cont

Gram staining and catalase test for all isolated

bacteria.

After initial selection microorganisms chosen fortheir ability to generate fermented milk with high ACEI

activity.

ACEI activity higher than 70% of 50% diluted wheyfraction was also a selection criteria.

Last selection criteria was based on highestinhibitory activity and absence of IPP and VPP peptides.


8/31

Isolation of microorganisms:-

Selected bovine milk aliquots MRS/M17 agar

of different sources

Incubation (48 h at 42 C/ 30 C)Isolation of the different colonies was performed by consecutive re-inoculation.

Preparation of fermented milk:-

Skim milk [10% (w/w) SMP]

sterilization (100 C / 20 min)


9/31

Inoculated with corresponding stock culture(105107 colony forming units (CFU)/ml)

overnight Incubation (30 or 42 C)

Pre-cultures

(3%, v/v)

sterile skim milk [10% (w/w) SMP]

Incubation (48 h/30 or 42 C)

Pasteurization (75 C / 1 min)


10/31

Assay of ACEI activity

Fermented milk aliquots

Vigorously stirred & centrifuged (20000 rpm for 10 min)

Supernatant

Filteration (whatman no 40 filter)Spectrophotometricassay:-100 L buffered substrate solution + 40 L whey fraction + 20 L ACEsolution

The reaction was carried out at 37 C for 30 min and stopped by theaddition of 1 m HCl

The hippuric acid released was extracted with 1 mL of ethyl acetate


11/31

vigorous stirring /20 s

samples were centrifuged (1500 rpm / 10 min)

750 L of organic phase was transferred to a glass tube

Evaporation (100C/25 min)

Residue + 800 L DW

Absorbance measurement at 228 nm(OD)

ACEI activity:-

% Inhibitory activity = ( A C ) / ( A B )

A = optical density without whey fraction.B = optical density without ACE.C = optical density with both.


12/31


13/31

Measurment of arterial blood pressure

Charles river lab.

SHR

WKY

Age: 17-20 week.

Wt: 300-350 g.

Sex: male

Kept at 23 C.

Single dose :- 5 ml/kg1.5 ml whey fraction/rat.

1.5 ml water

1.5 ml captoprilSBP & DBP measured by tail cuff

method before injection and

after 2,4,6,8 & 24 h postadministration

LE 5001, Instrument was used Rats were slightly anaesthetized

-Ve

SHR

SHR

+Ve


14/31

Results

I. Selection of microorganisms with ACEI activity

Statistical analysis:-Data expressed as mean values S.E.M for a minimum ofsix experiments, analyzed by Cluster analysis.A statistical program for windows was used for dataprocessing.

Initially isolated bacteria

231 Gram +Ve and catalase +VeMicroorganisms were selected.

46 strains of >70% ACEI

Activity were selected.


15/31

Enterococcus faecalis LP08 (CECT 5727) 30 4.47 100

LP11 (CECT 5728) 30 4.27 100

LP06 (CECT 5826) 30 4.62 98.2

LP10 (CECT 5827) 30 4.60 99.5

Lactococcus lactis subsp. cremoris LP25 30 5.54 91.0

LP26 30 5.64 92.8

LP27 30 5.67 91.4

Lactobacillus helveticus LP01 42 3.46 71.2

LP02 42 3.59 84.8

LP03 42 3.24 87.4

Lactobacillus brevis LP13 42 5.39 88.8

Microorganism strain temp pH ACEI (%)

7 strains Correspond to cocci

Lactobacilli predominant (43 strains)


16/31

Lactobacillus fermentum LP39 42 5.78 100

LP42 42 4.16 76.9

Lactobacillus rhamnosus LP07 42 3.58 74.7 LP09 42 3.63 72.7

LP17 42 4.49 80.0

LP19 42 4.78 79.7

LP23 42 4.47 78.2

LP30 42 4.62 94.1

LP31 42 4.85 84.4

LP32 42 4.62 89.1

LP33 42 4.89 78.6

LP34 42 4.72 80.4

LP35 42 5.04 99.0

LP37 42 5.63 83.9

LP38 42 5.31 85.3

LP40 42 5.14 78.0

LP41 42 4.50 85.5

LP43 42 4.47 91.4

LP44 42 5.44 85.6

LP45 42 5.81 95.9

LP46 42 5.23 84.5

LP48 42 4.17 88.8

LP50 42 5.71 93.4



17/31

Lactobacillus paracaseisubsp.paracasei LP12 42 5.51 91.8

LP14 42 5.69 99.7

Lactobacillus paracasei paracasei LP47 42 3.57 80.0

Lactobacillus acidophilus LP16 42 5.44 89.1

LP18 42 3.56 71.4

LP20 42 3.49 80.2

LP21 42 5.27 94.3

LP22 42 3.80 90.0

LP24 42 3.84 75.3

LP28 42 5.43 93.2

LP29 42 5.48 95.5

LP36 42 5.19 87.4



18/31

Cont.

Out of 46 LAB, 15 were studied in greater detail.ACEI activity was calculated as IC50 ,expressed in volume and in

protein concentration.

IC50

(L) = volume of whey fraction needed to give 50%

inhibition.= measure of pharmacological potency.= lower the value higher the ACEI potency.

Protein conc. of whey fraction determined by Kjeldahl method.

Cluster analysis variables:-pH, protein content & ACEI activity.


19/31

Fig. I Dendrogram of the 15 fermented milk products obtained byapplying cluster analysis to the data corresponding to pH, protein

content and ACEI activity.


20/31

Group I (high ACEI activity)

Enterococcus faecalis LP08(CECT 5727) 0.25 24.75 39

Enterococcus faecalis LP11 (CECT 5728) 0.38 24.79 59

Enterococcus faecalis LP06(CECT 5826) 0.30 25.87 49

Enterococcus faecalis LP10 (CECT 5827) 0.20 26.89 34

Lactobacillus helveticus LP01 1.96 7.73 95 +++ +++

Group II (moderate ACEI activity)

Lactobacillus rhamnosus LP17 2.35 11.95 175 +


Lactobacillus rhamnosus LP23 2.56 12.65 202

Lactobacillus acidophilus LP18 0.78 17.92 88

Lactobacillus acidophilus LP20 0.83 15.24 89 ++

Lactobacillus acidophilus LP28 2.39 11.93 178

Group III (low ACEI activity)

Lactococcus lactis subsp. cremoris LP25 7.89 10.53 520


Lactobacillus acidophilus LP22 7.13 8.59 383 +

Lactobacillus acidophilus LP24 9.15 6.81 389 + +

Microorganism IC50

Protein) IC50

IPP VPP

(L) (mg/ m) (g/ ml)


21/31

Cont

On the basis of IC50

values results clearly pointing out theE. faecalis strains

showing higher ACEI activity than the rest of bacteria.

IPP and VPP , were isolated from the milk fermented byLactobacillus

helveticus.

whey fractions of fermented milk samples were analyzed by HPLC-MS to

investigate the presence of these peptides.

The sample fermented withLb. helveticus LP01 contained both

antihypertensive peptides, IPP and VPP, and the ACEI activity of this

sample can probably be attributed to these peptides.

However, IPP and VPP were absent from the milk samples fermented with

different strains ofE. faecalis.

In vitro ACEI activity of fermented milk samples


22/31

Fig. II Doseresponse curves for the fermented milk samples produced by

several lactic acid bacteria strains. Values are the average of two different

assays.

E. faecalis

In vitro ACEI activity of fermented milk samples


23/31

Antihypertensive activity

Charles river lab.

SHR

WKY

1.5 ml whey fraction

1.5 ml water

1.5 ml captopril

+Ve

-Ve

SHR

SHR

1.5 ml whey fraction

SHR WKY

SBP 2483.67 172 4.74

DBP 2193.02 123 4.69

-Ve +Ve

SBP no effect clear decrease

DBP no effect clear decrease

Before injection:-After injection:-


24/31

Fig. III Decrease in SBP and DBP caused in spontaneously hypertensive ratsby the administration of water (), Captopril (50 mg kg1) (), and different milk

samples fermented by selectedE. faecalis strains.

CECT 5727 ()

CECT 5728 ()

CECT 5826 ( )

CECT 5827 ( ).


25/31

cont maximal decreases in SBP & DBP observed 6 and 4 h after theadministration of Captopril. fermented milk samples also caused a clear decrease in SBP &in DBP of the SHR.

The decrease in the DBP caused by the fermented milk samplesand 50 mg kg1 of Captopril were very similar.

Nevertheless, effect on SBP was less pronounced than causedby this dose of drug.

Captopril produced a maximum decrease in the SBP 6 h after

administration, but the maximal effect of the fermented milksamples was observed after 4 h.

In addition, the effect of all these fermented milk samples onthe SBP, as well as on the DBP, were very similar. These effects

reverted 24 h after the administration.


26/31

Fig. IV Decrease in SBP and DBP caused in Wistar-Kyoto rats by theadministration of water (), Captopril (50 mg kg1) (), and different

milk samples fermented by selectedE. faecalis strains.

CECT 5727 ()

CECT 5728 ()

CECT 5826 ( )

CECT 5827 ( ).

Fig. shows that none of

the administeredproducts modified the

SBP or the DBP of the

normotensive WKY rats.


27/31

Cont


28/31

Cont In this study, all experiments were performed with pasteurizedfermented milk in order to avoid the presence of viable bacteriain the final product.

Enterococcusstrains used in this study were isolated from rawmilk and the pathogenic potential of Enterococcusstrains seemsto be related to their origin.

Some clinical isolates of Enterococcusare pathogenic in

contrast to food isolates, which normally do not present anyvirulent factors.

In fact, several food products containing these bacteria havea long history of consumption without health risks.

Individual examination of these bacteria is in progress toensure the safety of our strains. Milk fermented by E. faecalisshowed the lowest IC50 values in

this study (3459 g mL1

).


29/31

cont

clear antihypertensive effect observed when the fermented milksamples were administered to SHR by gastric intubation.

Maximal decrease in SBP and DBP was always attained 4 h afteradministration and it returned to baseline after 24 h.

Results indicate that the ACEI peptides present in fermentedmilk samples can be efficiently absorbed through the intestine of

the animals in an active form.

It is also important to point out that the administration of thefermented milk samples did not change the arterial blood pressureof the normotensive WKY rats.

It suggest that the effect of the fermented milk samples isspecific to hypertensive state.

Therefore, these products could be used as functional foodswithout any risk in normotensive subjects.


30/31

Results suggest that raw cows milk is an excellent source ofwild lactic acid bacteria able to produce fermented milk withACEI activity.

Four selected strains of E. faecaliswere able to producepasteurized fermented milk with potent ACEI andantihypertensive activity.

Further studies to identify the peptides responsible for theseactivities are in progress.

This study raises the possibility of using E. faecalisstrains asstarters to obtain functional antihypertensive products.

Conclusion


31/31

anti hypertensive pro bio tics

Documents