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Applied and Environmental Microbiology, July 2002, p. 3566-3569, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3566-3569.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cardiovascular Effects of Fermented Milk Containing Angiotensin-Converting Enzyme Inhibitors Evaluated in Permanently Catheterized, Spontaneously Hypertensive Rats

Anders Fuglsang,^1* Dan Nilsson,² and Niels C. B. Nyborg³

Royal Danish School of Pharmacy, Institute of Pharmacology, 2100 Copenhagen Ø,¹ Novo Nordisk A/S, 2670 Måløv,² Chr. Hansen A/S, 2970 Hørsholm, Denmark³

Received 5 December 2001/ Accepted 19 April 2002

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In this study, two strains of Lactobacillus helveticus were used to produce fermented milk rich in angiotensin-converting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of L. helveticus gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin-converting enzyme (EC 3.4.15.1) (ACE) is known to play a role in the regulation of blood pressure in animals, including humans. The enzyme converts angiotensin I into angiotensin II, the former being an inert peptide and the latter being a pressor agent. The enzyme is also responsible for the breakdown of bradykinin, which is a dilatory peptide. The enzyme is thus an obvious drug target in treatment of certain cardiovascular diseases, including hypertension. Lactic acid bacteria are known to produce inhibitors of the enzyme in various amounts during fermentation (5, 10, 13). The inhibitors are formed by the bacterial proteinases when the lactic acid bacteria hydrolyze milk proteins, mainly casein, into peptides, which can be used as nitrogen sources necessary for growth. All of the ACE inhibitors known to date that are formed in milk during fermentation are peptides that act as competitive inhibitors, such as Ile-Pro-Pro and Val-Pro-Pro (11). In some cases it has been demonstrated that fermented milk rich in inhibitory substances can lower systolic blood pressure in spontaneously hypertensive rats (SHR) after oral administration (11, 13, 14).

Since the inhibitors formed are peptides, which are formed by degradation of caseins, proteolysis plays an important role. In theory, both specificity and overall proteolytic activity may have a significant role in the formation. The fact that the proteolytic systems of lactic acid bacteria are quite unspecific, i.e., that they cleave, for instance, caseins at a high number of places (for a review, see reference 8), combined with the fact that ACE can be inhibited by many different peptidic structures (for instance, Val-Pro-Pro [11], Ile-Tyr [15], and Lys-Val-Leu-Pro-Val-Pro [9], all of which inhibit ACE in the micromolar range), led to the investigation of a possible correlation between crude proteolysis (measured as free amino groups) and ACE inhibition. It was shown that, in general, the higher the number of free amino groups formed during fermentation, the higher the chance that the same fermented substance will be able to inhibit ACE significantly, even though this rule is not always strictly obeyed. Among the strains showing a relatively high amount of free amino group formation, two strains capable of producing milk with high activity in vitro against ACE were identified and were shown to be able to decrease the in vivo activity of circulatory ACE in rats, measured as a decreased ability to convert angiotensin I to angiotensin II after oral administration of the fermented milk (3). This study did therefore not involve measurements of blood pressure per se; rather, it quantified pressure increases in response to injected angiotensin I. Since the ACE inhibitors in modern use against hypertension are able to decrease this pressure response to angiotensin I injection, it seems relevant to proceed with a more specific study on the effect of the fermented substances on blood pressure.

The aim of this study was to use permanently catheterized rats to investigate if the fermented milks, i.e., those that are able to inhibit circulatory ACE in vivo as mentioned before, are also able to decrease blood pressure in SHR measured through a permanent aortic catheter. This technique, in combination with a data acquisition system, also offers the advantage of being able to extract heart rates from the pulsatile signal. Since the mean arterial pressure (MAP) is roughly given by the product of stroke volume, peripheral resistance, and heart rate, the latter is an important parameter in the regulation of blood pressure.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria and fermentations.
Lactobacillus helveticus CHCC637 and L. helveticus CHCC641 were propagated in heat-treated 9.5% reconstituted skim milk, and fermentations were carried out at 37°C overnight, using a 1% inoculum. Lactococcus lactis CHCC1448 was propagated similarly but at 30°C. The three strains were obtained from the Chr. Hansen Culture Collection (Chr. Hansen A/S, Hørsholm, Denmark). All chemicals and reagents were from Sigma unless otherwise stated.

Measurement of in vitro ACE inhibition.
The method of Cushman and Cheung (2) was used with some modification. Fermented milk was centrifuged at 4°C in an Eppendorf 5804R centrifuge (Eppendorf GmbH, Hamburg, Germany) at 4,500 rpm. The whey supernatant was adjusted to pH 8.3 with 10 N NaOH and was given 14,000 rpm in a bench centrifuge (157MP; Ole Dich, Hvidovre, Denmark) for 10 min. The supernatant was used in the assay where 80 µl was mixed with 200 µl of substrate buffer (0.3 M NaCl, 0.1 M boric acid, and 5 mM hippuryl-histidyl-leucine, pH 8.3) and 20 µl of ACE solution (rabbit lung ACE; 0.1 U/ml). The mixture was kept at 37°C for 60 min, and 250 µl of 1 N HCl was added to stop the reaction. The hippuric acid formed was extracted with 1,700 µl of ethyl acetate. Fourteen hundred microliters of the organic phase was transferred to a heat block at 95°C for 15 min to evaporate all ethyl acetate. The residues were then resuspended in 2 ml of water and were measured at 214 nm with a Secomam Anthelie spectrophotometer (Secomam, Ales, France) using quartz cuvettes. We determined 100% activity by using 80 µl of substrate-free buffer instead of sample, and 0% activity was determined using 20 µl of water instead of enzyme solution. Unfermented milk cannot readily be analyzed this way but was acidified to pH 3.75 and processed as described above.

Measurement of peptide content.
The whey peptide content was quantified using a modified version of the method by Church et al. (1). One hundred milliliters of reagent was prepared by mixing 50 ml of 0.1 M borax, 5 ml of 20% (wt/vol) sodium dodecyl sulfate, 540 µl of thiolactic acid (Aldrich Chemicals), and 2.5 ml of o-phthaldialdehyde solution (50 mg/ml in 96% ethanol) and adjusting the volume to 100 ml with water. Twenty-five microliters of test solution diluted two- or threefold was mixed with 1,000 µl of reagent, and the reaction mixture was measured at 340 nm, with the reagent as reference. Since no obvious peptide standards exist for the peptide mixtures formed by lactic acid bacteria, the peptide content was quantified using both casein tryptone (Difco Laboratories, Detroit, Mich.) and casein peptone (Organotechnie, La Courneuve, France) as standards. Both peptone and tryptone yielded linear standard curves in the range of 0 to 2.5 mg/ml (r² > 0.999).

Permanent catheterization of rats.
Twelve-week-old male SHR were obtained from Møllegaard & Bomholtgård A/S, Ejby, Denmark, and had free access to tap water and standard Altromin pellet food. Lights were on from 6 a.m. to 6 p.m. The rats were allowed to acclimatize at least 7 days before being used in the experiment. Under narcolept anesthesia with midazolam (Alpharma, Oslo, Norway) and fluanisone/fentanyl (Hypnorm; Janssen, Beersen, Belgium), the rats had tygon tubing (Copenhagen University, Institute of Pharmacology, Copenhagen, Denmark) inserted in the aorta through the left carotid artery. The distal end of the tubing protruded from the neck. The rats were given 3 days of postoperative pain treatment by subcutaneous meloxicam injections (Metacam Vet; Boehringer Ingelheim GmbH, Ingelheim, Germany). The catheters were flushed with saline at up to 48-h intervals in order to keep them free of clots and were sealed with a fluid consisting of glucose (50%), heparin (500 U/ml; Leo Pharmaceuticals, Ballerup, Denmark), and streptokinase (10,000 U/ml; Aventis-Behring, Danderyd, Sweden).

On the day of the experiment, the rats were placed in a restrainer and the external end of the catheter was flushed with saline and connected to a pressure transducer (P23XL; SpectraMed, Bilthoven, The Netherlands) for recording of MAP. The pulsatile signal was recorded using a DAQ801/DaqEz data acquisition system (Quatech Inc., Ohio) running at a 50-Hz sampling rate. After recording of their baseline MAP and heart rate, the rats were administered fermented milk (10 ml/kg of body weight) by gavage and put back in the cage. Four hours after the feeding they were put into the restrainer again and had their MAP continuously measured for 4 h. As a negative control, unfermented milk was used along with milk fermented by CHCC1448 (the latter being a strain that produces fermented milk with very little ACE inhibition). This control more closely resembles fermented milk and did not cause significant ACE inhibition in vivo in a previous study.

Data analysis.
BeePee v2 (a 32-bit Windows application programmed by Anders Fuglsang) was used to extract data for the heart rate and MAP from the data acquisition files. GraphPad Prism 2.01 (GraphPad Inc.) was used to perform two-way analysis of variance on the blood pressure and heart rate data. This program was also used to fit data for the inhibition in vitro to the Michaelis-Menten equation and thus to obtain concentration-response curves and 50% inhibitory concentrations (IC₅₀s). A P of <0.05 was considered statistically significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Figure 1 shows the dose-response curves of in vitro ACE inhibition of the four types of milk used in the study. The two strains of L. helveticus clearly produce fermented milk with ACE-inhibitory properties. The data fit with a maximal effect of approximately 100% inhibition, which indicates that the mode of inhibition is competitive. The ACE-inhibitory IC₅₀s (indicators of potency towards the enzyme) are listed in Table 1 along with data for optical density at 600 nm, peptide content, and pH. Note that the IC₅₀ given in microliters signifies the volume that inhibits the enzyme by 50% under assay conditions where the total volume is 300 µl and is therefore a measure of the concentration-dependent pharmacological property called potency. The IC₅₀ given in milligrams per milliliter tells how many milligrams of the peptide pool per milliliter are needed to inhibit ACE by 50% and is therefore an indicator of how specific the obtained peptide pool is against ACE, regardless of whether or not the peptides are present in large or small amounts (4, 7). Figure 2 shows the plot of MAP versus time, where MAP is the initial MAP minus the MAP at a specific time point. Figure 3 shows the change in heart rate calculated the same way. Graphically, the curves for the test groups display lower MAP than do the curves for their controls. Both the blood pressures and heart rates are statistically significantly different from those for the controls, as detailed in Table 2. The reason why differences are shown rather than actual values is that there was a large variability between the individual blood pressures and heart rates among the rats.

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FIG. 1. Dose-response curves (effect as function of the logarithm of the dose in microliters) for the four types of milk used in this study. The lines depict the best fit curves according to the Michaelis-Menten equation. Note that unfermented milk had to be acidified with lactic acid before construction of this kind of curve was possible. Data are means of two replicates. Error bars represent minima and maxima. White circles, L. helveticus CHCC637; black circles, L. helveticus CHCC641; black triangles, L. lactis CHCC1448; and white triangles, unfermented milk.

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TABLE 1. Comparison of milk and bacterial strainsa

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FIG. 2. The MAP effects of milk fermented with L. helveticus CHCC641 (boxes, n = 5) (a) and L. helveticus CHCC637 (boxes, n = 6) (b) plus those of their controls, L. lactis CHCC1448 (triangles, n = 5) and unfermented milk (circles, n = 6). Data are represented as values relative to the initial baseline blood pressure. The time scale is relative to the time of feeding. Error bars represent standard errors of the means.

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FIG. 3. The heart rate effects of milk fermented with L. helveticus CHCC641 (boxes, n = 5) (a) and L. helveticus CHCC637 (boxes, n = 6) (b) plus those of their controls, L. lactis CHCC1448 (triangles, n = 5) and unfermented milk (circles, n = 6). Data are represented as values relative to the initial baseline blood pressure. The time scale is relative to the time of feeding. Error bars represent standard errors of the means. HR (bpm), change in heart rate (beats per minute).

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TABLE 2. Comparison of two L. helveticus strainsa

Top Abstract Introduction Materials and Methods Results Discussion References

 
The two strains of L. helveticus produce competitive inhibitors (maximum inhibition according to the fit is approximately 100%), which is in accordance with earlier reports on the mode of action for milk-derived inhibitors of ACE. The peptide content, pH, and optical density resemble those previously obtained with other bacteria (13).

The peptide content, though difficult to interpret directly because of the lack of a standard that reflects the real distribution of peptidic molecular weights of the peptides liberated by the action of bacterial proteinases, was lowest in the two controls as expected. Calculation of IC₅₀ in milligrams per milliliter yields a number that is characteristic of the peptide pool formed and is thus an indicator of the amount of peptide pool necessary to inhibit ACE by 50% under the present assay conditions. In theory, differences in the peptide pools might therefore be detected by this value. Using nonparametric statistics could be a way of detecting differences in proteolytic specificity among strains. This, however, would call for a higher number of observations than present in this study. The data for optical density, pH, and peptide content are similar to those in previous reports on a strain of L. helveticus, designated CP790, that had the ability to produce inhibitors (11). Milk fermented with this strain proved highly hypotensive in a tail cuff study, with a drop of up to about 30 mm Hg in systolic blood pressure in SHR (11, 13). As blood pressure-lowering agents do not change MAP and systolic pressure equally, the data presented here cannot be directly compared to those from reports on systolic effects. It is interesting, however, that we detect significant effects on the heart rate. It is well known that drugs used in the treatment of hypertension may affect heart rate; ß-adrenergic antagonist (e.g., propranolol) blocks the sympathetic action of the ß-receptors on the heart and thus decreases the heart rate directly. In contrast to this, calcium channel blockers of the dihydropyridine type (e.g., felodipine) cause a compensatory stimulation of the sympathetic nervous system, thereby raising the heart rate. As angiotensin II raises arterial tone, thereby raising blood pressure, one could argue that ACE inhibition could evoke a reflex-mediated increase in the heart rate, but since angiotensin II activates the sympathetic nervous system by presynaptic facilitation (12), one could also argue that inhibition of ACE in vivo should decrease the heart rate. Even though receptors for angiotensin II are present in both cardiac atria and ventricles throughout the life cycle (6), the high number of other endogenous regulatory and compensatory pathways makes it difficult to predict what effects on the heart rate ACE inhibitors should have, especially when we do not know if the active substances affect other targets. The present results therefore allow us only to conclude that the antihypertensive effects observed with ACE-inhibitory fermented milk may be mediated by a decreased heart rate but do not absolutely prevent us from excluding dilatory effect in the periphery. Regional flow studies may be a way of assessing such effects.

From Table 2 it is also clear that the fermented milk produced by L. helveticus CHCC637 and L. helveticus CHCC641 does not yield identical probability values versus the two controls. The reason for this could well be differences in the pool or potency of the obtained sample, as Table 1 suggests. It is also worth noting that the heart rate effect was insignificant versus that of unfermented milk but significant versus that of the L. lactis CHCC1448 product. The reason must be differences in the chemical composition of these two controls, such as differences in pH or peptide content.

The data in this study do not answer some very fundamental questions, such as when the effects are most pronounced or how long the duration of action is. It is not within our present permission to measure the animals outside the interval of 4 to 8 h after oral administration. During the 4 h in the restrainer when MAP and the heart rate are being measured, the animals do not have access to water or food. This situation is far from real life, and so the data presented may not accurately reflect the pharmacological potential of the samples. This problem could be overcome in the future by using radiotelemetry.

All blood pressure curves (both tests and controls) seem to decrease with time, which may be explained by the fact that the animals lose body fluid during the 4 h. Another possibility is that the rats gradually acclimatize to being in the experiment over these 4 h. In that case, the initial blood pressures measured here do not reflect the factual baseline blood pressures, and the significant figures could instead indicate an altered ability to acclimatize during acute stress (as imposed by the experimental procedure).

An obvious future objective is to evaluate the two types of fermented milk in hypertensive animals using both tail cuffs and radiotelemetry. Studies on the plasma half-lives of ACE-inhibitory, milk-derived peptides are under way in order to elucidate and characterize the true mechanism of action of antihypertensive peptides formed during milk fermentation.

 
This work was supported by the Danish Heart Foundation, grant number 01-1-2-19-22-893. The Danish Animal Experimentation Inspectorate approved the use of permanently catheterized rats for this study.

We thank Dennis Neal (Rpm-Psi Inc., Northridge, Calif.) for competent help during the data acquisition setup. Special thanks go to Henrik Holm-Kjar (Zealand Pharmaceuticals A/S, Glostrup, Denmark), who taught us the in vivo technique.

 
* Corresponding author. Mailing address: Royal Danish School of Pharmacy, Institute of Pharmacology, Universitetsparken 2, 2100 Copenhagen Ø, Denmark. Phone 45 35306000. Fax: 45 35306020. E-mail: anfu{at}dfh.dk.

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Applied and Environmental Microbiology, July 2002, p. 3566-3569, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3566-3569.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fuglsang, A. Articles by Nyborg, N. C. B. Search for Related Content PubMed PubMed Citation Articles by Fuglsang, A. Articles by Nyborg, N. C. B. Agricola Articles by Fuglsang, A. Articles by Nyborg, N. C. B.