Antihypertensive activity of milk

fermented by Enterococcus faecalis strains

isolated from raw milk

RAJESH KUMAR
Ph.D 1st yr
Dairy Microbiology Division
N.D.R.I
 Introduction
• Hypertension :- Cardiovascular diseases.

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

(Ondetti et al, 1977)

Angiotensin I ACE
Angiotensin II
(inactive decapeptide) (active octapeptide)

Active Bradykinin inactive Bradykinin

X
Vasodilation Nov 15,2005.
Cont….

 Inhibition of ACE generate Antihypertensive

effect.

 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

• Lactibacillus helveticus
• Lb. delbrueckii subsp. bulgaricus.

 Milk fermented with Lb. helveticus LBK-16h has

demonstrated significant antihypertensive effects
in humans.
Seppo et al, 2002; Seppo et al, 2003.
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 and

characterized.
 AIM

To select wild strains of bacteria from raw cow’s

milk which were able to produce fermented milk with
inhibitory ACE activity.

 Antihypertensive effect of fermented milk

produced with four selected strains of E. faecalis on
the arterial blood pressure of SHR and normotensive
Wistar-Kyoto rats (WKY).
 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 microorganism

isolation.
Cont…

 Gram staining and catalase test for all isolated

bacteria.

 After initial selection microorganisms chosen for

their ability to generate fermented milk with high ACEI
activity.

 ACEI activity higher than 70% of 50% diluted whey

fraction was also a selection criteria.

 Last selection criteria was based on highest

inhibitory activity and absence of IPP and VPP peptides.
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)

Inoculated with corresponding stock culture
(105–107 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)

Assay of ACEI activity
Fermented milk aliquots

Vigorously stirred & centrifuged (20000 rpm for 10 min)

Supernatant

Filteration (whatman no 40 filter)

Spectrophotometric assay:-

100 μL buffered substrate solution + 40 μL whey fraction + 20 μL ACE

solution

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

The hippuric acid released was extracted with 1 mL of ethyl acetate
 vigorous stirring /20 s

samples were centrifuged (1500 rpm / 10 min)

750 μL of organic phase was transferred to a glass tube

Evaporation (100°C/25 min)

Residue + 800 μL 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.
Analysis by on-line RP-HPLC-MS/MS

Reversed-phase high-performance liquid chromatography separation of

supernatants were performed on an Agilent HPLC system connected on line
to an Esquire-LC quadrupole ion trap instrument.

 250 mm×4.6 mm Widepore C18 column were used.

 50 μL = injection volume.

 Bruker Daltonik (version 2.1) used to process the MS spectra and to

perform peptide sequencing.

Peptides IPP and VPP prepared by a conventional Fmoc solid-

phase synthesis method with a 431A peptide synthesizer.

• Purity verified by analytical RP-HPLC coupled to MS.

 Measurment of arterial blood pressure
Single dose :- 5 ml/kg 1.5 ml water
1.5 ml whey fraction/rat.

-Ve
Age: 17-20 week.
SHR Wt: 300-350 g.
Charles river lab. Sex: male
Kept at 23 °C. +Ve SHR

WKY SHR

SBP & DBP measured by tail cuff

method before injection and 1.5 ml captopril
after 2,4,6,8 & 24 h post
administration

LE 5001, Instrument was used Rats were slightly anaesthetized

Statistical analysis:-
Data expressed as mean values ± S.E.M for a minimum of
six experiments, analyzed by Cluster analysis.
A statistical program for windows was used for data
processing.

Results
I. Selection of microorganisms with ACEI activity

Initially isolated bacteria

231 Gram +Ve and catalase +Ve

Microorganisms were selected.

46 strains of >70% ACEI

Activity were selected.
 Microorganism strain temp pH ACEI (%)
Enterococcus faecalis LP08 (CECT 5727) 30 4.47 100

LP11 (CECT 5728) 30 4.27 100

7 strains Correspond to cocci

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

Lactobacilli predominant (43 strains)
LP03 42 3.24 87.4

Lactobacillus brevis LP13 42 5.39 88.8

Microorganism strain temp pH ACEI (%)

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
 Microorganism strain temp pH ACEI (%)
Lactobacillus paracasei subsp. 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

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 (μL) = 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.
Fig. I  Dendrogram of the 15 fermented milk products obtained by
applying cluster analysis to the data corresponding to pH, protein
content and ACEI activity.
 Microorganism IC50 Protein) IC50 IPP VPP
(μL) (mg/ m) (μg/ ml)
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 LP19 2.77 11.21 194 + −

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 rhamnosus LP09 10.71 9.11 609 + −

Lactobacillus acidophilus LP22 7.13 8.59 383 + −

Lactobacillus acidophilus LP24 9.15 6.81 389 + +

Cont…
On the basis of IC50 values results clearly pointing out the E. faecalis strains
showing higher ACEI activity than the rest of bacteria.

IPP and VPP , were isolated from the milk fermented by Lactobacillus
helveticus.

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

investigate the presence of these peptides.

The sample fermented with Lb. 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 of E. faecalis.
 In vitro ACEI activity of fermented milk samples

E. faecalis

Fig. II Dose–response curves for the fermented milk samples produced by
several lactic acid bacteria strains. Values are the average of two different
assays.
 Antihypertensive activity

1.5 ml water
1.5 ml whey fraction
-Ve

SHR
Charles river lab. SHR
+Ve
1.5 ml whey fraction

WKY SHR

Before injection:- After injection:-

1.5 ml captopril
SHR WKY -Ve +Ve
SBP 248±3.67 172± 4.74 SBP no effect clear decrease

DBP 219±3.02 123± 4.69 DBP no effect clear decrease

 CECT 5727 (●)
CECT 5728 (■)
CECT 5826 ( )
CECT 5827 ( ).

Fig. III Decrease in SBP and DBP caused in spontaneously hypertensive rats
by the administration of water (×), Captopril (50 mg kg−1) (–), and different milk
samples fermented by selected E. faecalis strains.
 cont…
 maximal decreases in SBP & DBP observed 6 and 4 h after the
administration 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 samples

and 50 mg kg−1 of Captopril were very similar.

 Nevertheless, effect on SBP was less pronounced than caused

by this dose of drug.

Captopril produced a maximum decrease in the SBP 6 h after

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

 In addition, the effect of all these fermented milk samples on

the SBP, as well as on the DBP, were very similar. These effects
reverted 24 h after the administration.
 CECT 5727 (●)
CECT 5728 (■)
CECT 5826 ( )
CECT 5827 ( ).

Fig. shows that none of

the administered
products modified the
SBP or the DBP of the
normotensive WKY rats.

Fig. IV  Decrease in SBP and DBP caused in Wistar-Kyoto rats by the

administration of water (×), Captopril (50 mg kg−1) (−), and different
milk samples fermented by selected E. faecalis strains.
 Discussion
 Our results show that a wide range of lactobacilli are able to
produce ACEI peptides during milk fermentation. To date, only
Lb. helveticus and Lb. bulgaricus had demonstrated this capacity.

 Identification of Enterococcus strains as producers of fermented

milk with ACEI and antihypertensive activity is the most remarkable
finding.

 The possibility of using these bacteria as producers of bioactive

peptides has not been considered although the proteolytic activity of
these bacteria is well known.

 Some Enterococcus strains constitute part of the natural

microflora in many dairy products, especially cheese.

 Use of E. faecalis in a food system is still a matter of

controversy due to their pathogenic potential.
Cont…
 In this study, all experiments were performed with pasteurized
fermented milk in order to avoid the presence of viable bacteria
in the final product.

 Enterococcus strains used in this study were isolated from raw

milk and the pathogenic potential of Enterococcus strains seems
to be related to their origin.

 Some clinical isolates of Enterococcus are pathogenic in

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

 In fact, several food products containing these bacteria have

a long history of consumption without health risks.

 Individual examination of these bacteria is in progress to

ensure the safety of our strains.

 Milk fermented by E. faecalis showed the lowest IC50 values in

this study (34–59 μg mL−1).
cont…
 clear antihypertensive effect observed when the fermented milk
samples were administered to SHR by gastric intubation.

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

 Results indicate that the ACEI peptides present in fermented

milk 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 the

fermented milk samples did not change the arterial blood pressure
of the normotensive WKY rats.

 It suggest that the effect of the fermented milk samples is

specific to hypertensive state.

 Therefore, these products could be used as functional foods

without any risk in normotensive subjects.
 Conclusion

•Results suggest that raw cows’ milk is an excellent source of

wild lactic acid bacteria able to produce fermented milk with
ACEI activity.

•Four selected strains of E. faecalis were able to produce

pasteurized fermented milk with potent ACEI and
antihypertensive activity.

•Further studies to identify the peptides responsible for these

activities are in progress.

•This study raises the possibility of using E. faecalis strains as

starters to obtain functional antihypertensive products.
THANKS