Antihypertensive Effect of Milk Fermented by Lactiplantibacillus plantarum K79 on Spontaneously Hypertensive Rats

L. plantarum K79 was isolated from kimchi. This strain was selected after screening it for probiotic properties such as acid and bile tolerance, antibacterial activity, antibiotic tolerance and ACE inhibitory activity. The culture was maintained in an MRS broth (Difco, New York, NY, USA), and the patent accession number is KACC 81222BP.

Skim milk powder (13.5%) and 1% glucose were mixed, sterilized at 90°C for 5 min, and then cooled to 37°C. At this time, lyophilized L. plantarum K79 strain activated for 1 h was inoculated and cultured with stirring at 37°C until the pH reached 4.2. The culture was heat-treated by stirring at 85°C for 20 min, cooled to below 50°C, and then spray-dried and powdered. The powdered raw materials were suspended and used for testing.

The experiment included control groups composed of 5-wk-old SHR (SLC, Tokyo, Japan) male rats, and normal groups (NGs) composed of Wistar-Kyoto rats (WKY), which were used after 1 wk of acclimatization. The experimental animals were fed with the AIN-93G diet, and during the acclimatization period the filtered drinking water was changed every day so that it could be consumed freely. During the breeding period, the temperature was 23±1°C, humidity was 50±5%, noise was less than 60 phones, lighting time was 08:00–20:00 (12 h a day), illuminance was 150–300 Lux, ventilation was 10–12 times per hour. This experiment was conducted with the approval of the Korea Food Research Institute (KFRI-M-22024) in compliance with the regulations on animal experimentation ethics. During the breeding period, the experimental animals were fed with regular solid feed (Samtako, Osan, Korea), and their blood pressure was measured at the end of the acclimatization period. Individual identification was indicated.

Experimental group: NG (WKY): distilled water, control group (SHR): distilled water, high treatment group (HTG; SHR): 500 mg/kg/day, medium treatment group (SHR): 335 mg/kg/day, low treatment group (SHR): 170 mg/kg/day, positive control group (PCG; SHR): Enalapril, 10 mg/kg/day. Each group used in the experiment consisted of 8 animals. At this time, the oral administration method, which is the clinical application route of this formulation, was selected and each sample was directly administered into the stomach of each experimental animal.

All the experimental animals were weighed once per week before the start of administration, after the start of administration, and until the end of the test. For each breeding box, the total amount of feeding and watering on the day and the remaining amount the next day were measured once per week for 4 wk after the start of administration, and the amount of daily consumption was indicated for each group. The systolic pressure of each rat was measured with a tail cuff blood pressure meter (INDIR, Model LE5002, Panlab, Barcelona, Spain), and was maintained at 32°C for 30 min to detect the pulse of each rat’s tail artery before measurement.

At the end of the 10-wk experimental period, blood samples were collected from all the rats and immediately placed in sterile tubes. Serum was collected by centrifugation at 2,000×g for 15 min at 4°C. The serum samples were then analyzed for total cholesterol (TC), triglyceride (TG) concentration, high density lipoprotein (HDL) cholesterol, and low density lipoprotein (LDL) cholesterol by GCCL (Yongin, Korea). Also, the liver, kidneys, lungs, heart and testes of the rats were quickly isolated after sacrifice and weighed.

The angiotensinogen (AGT) protein expression, angiotensin II protein, and renin protein content in the plasma were measured using a Rat AGT ELISA kit, a Rat Angiotensin II ELISA kit, and a Rat Renin ELISA kit (MyBioSource, San Diego, CA, USA), respectively.

According to the method of Averill et al. (2003), kidney tissue was left in 4% formalin for 48 h before being transferred to 70% ethanol. Blocks of cardiac tissue were imbedded in paraffin; 5 μm sections were transferred to slides and the paraffin was removed by sequential washes with xylene, 100% ethanol, 95% ethanol, 75% ethanol, and double-distilled water. Sections of tissue were incubated with 3% hydrogen peroxide for 5 min, washed with PBS (pH 7.2), dried, and then incubated with 5% normal goat serum for 1 h at room temperature. The sections were washed with PBS and incubated overnight at 4°C with an affinity-purified rabbit polyclonal antibody to angiotensin-1 at 1:25 dilution of the antibody in 1% BSA. The Angiotensin-1 antibody was purified. The next day, the tissues were washed with PBS and incubated for 3 h at 4°C with a biotinylated anti-rabbit antibody at a dilution of 1:400 in 1% BSA. The slides were rinsed with PBS, blotted dry, and reacted immunocytochemically by avidin-biotin solution and stained brown with 3,3′-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA) in Tris buffered saline (0.05 mol/L, pH 7.6 to 7.7). The reaction was stopped in PBS, and the sections were rinsed in double-distilled water before being counterstained with hematoxylin (Sigma-Aldrich). The tissue sections were dehydrated in ethanol (70% to 100%) and then Histoclear (National Diagnostics, Atlanta, GA, USA). Finally, they were mounted under coverslips with Histomount.

The results are expressed as the mean±SD per experimental group using SPSS version 23 (IBM, Armonk, NY, USA). The significance of the differences was analyzed by conducting a one-way analysis of variance (ANOVA) using Duncan’s multiple range tests. Significance was considered at p<0.05.

Looking at the change in weight according to the experimental period, there was a significant difference between the NG (WKY), which was fed with distilled water, and the hypertensive rat (SHR) group within the same period (Fig. 1). This was found to be consistent with Gattone’s (1986) report that WKY had a similar weight to SHR at birth, but that its weight became greater than that of SHR over time. However, there was no significant difference in average body weight between the treatment group and the control group, indicating that milk fermented by L. plantarum K79 used in the experiment did not have a significant effect on the weight of the rats.

Fig. 1. Change in body weight of SHR fed with experimental diet (g/rat) for 10 wk. All values are the mean±SD (n=8). ^* In the same column are significantly different from NG and the other group (p<0.05). NG, normal group (WKY) with distilled water; NCG, negative control group (SHR) with distilled water; PCG, positive control group (SHR) with Enalapril; LTG, low treatment group (SHR); MTG, medium treatment group (SHR); HTG, high treatment group (SHR); WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats.

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There was a slight difference in intake, but no significant change was observed, indicating that the fermented milk containing L. plantarum K79 had nothing to do with the weight gain of the rats. However, at 7 wk of feeding, the low-treatment group showed a significant increase compared to the normal and medium, high-treatment groups (Table 1).

The results of measuring the blood pressure according to the experimental period showed that there was a significant change in blood pressure in the NG, followed by the PCG, treatment group, and negative control group (NCG), until 10 wk. When comparing the treatment group and the NCG, the treatment group showed a tendency to lower blood pressure until 6 wk, but it was found that there was no significance (Table 2). However, after 8 wk, blood pressure was lowered more significantly in the HTG (209.9±13.3 mmHg) than in the NCG (230.8±7.3 mmHg). Ishiguro et al. (2012) reported that systolic blood pressure (SBP) and diastolic blood pressure were reduced by more than 30 mmHg and 20 mmHg, respectively, after oral administration of purified sweet potato protein hydrolyses to SHR. Tsai et al. (2006) found that the SBP of SHR decreased by 19 mmHg after 8 wk of oral administration of fermented soymilk with lactic acid bacteria. These results were similar to the results obtained in this study. According to Alshuniaber et al. (2021) and Yuan et al. (2022), this drop of blood pressure in rats is related to inflammation and could be obtained by inhibiting the ACE activity of the RAS.

Regarding blood lipids, TC, HDL cholesterol and LDL cholesterol were all significantly higher in the NG than in the hypertensive rat group, while TG was significantly lower in the NG (Table 3). This finding was consistent with the report of Cho et al. (2018), who observed that the SHR group had lower TC and HDL-C levels than the WKY group. Among the blood pressure rat groups, TC and LDL cholesterol were slightly higher in the medium-dose treatment group, but the levels of HDL cholesterol and TG were insignificant.

When examining the weight of each organ of the SHR euthanized 10 wk after administration of the fermented product, the weight of the liver in the PCG was found to be significantly higher than that in the NG, while the weight of the kidneys was significantly lower in the NG than in the other groups (Table 4). There was no significant change in the weight in the lungs of all groups, and the weight of the heart was significantly higher in the NCG and the treatment group than in the PCG and the NG. The organs of the NG weigh less than those of the other groups, but the testes weigh significantly more. This was consistent with the report by Walter and Hamet (1986) that the heart, kidneys and liver weighed less in WKY than in SHR.

As is well known, Ang II is produced systematically. AGT, a substrate of RAS, is released by the liver and broken down in the circulation to form Ang I by renin secreted by the proximal glomerular apparatus of the kidneys. Vasodilator I (Ang I) is then readily activated, and Ang II is caused by ACE, which is expressed at high levels primarily on the surface of endothelial cells in the pulmonary circulation. When the concentration of these biomarkers increases, it affects the increase in blood pressure (Ichihara et al., 2004; Yim and Yoo, 2008). Table 5 shows changes in blood pressure-related biomarkers in the plasma of rats after 10 wk. It was confirmed that the NG, PCG, and HTG had significantly lower AGT protein expression than the NCG. Angiotensin-II showed significantly lower expression in the NG, PCG, and treated groups than in the NCG. Renin also was expressed the least in NG compared to NCG, followed by PCG and HTG. Therefore, it is judged that significantly suppressing blood pressure-related biomarker protein expression had the effect of lowering the blood pressure of the treatment group.

Human angiotensin is expressed in the tissues of diverse organs including those of the liver, heart, blood vessel walls, brain, and kidneys, and in adipose tissue. Although all organ systems have certain components of the RAS, the kidneys have all the components of the RAS, and also show compartmentalization and intracellular accumulation of tubular and interstitial networks (Kobori et al., 2007)

Fig. 2 shows the results of an experiment confirming the expression of angiotensin-I using immunohistochemistry. Looking at the intrarenal glomeruli, it was found that the protein expression of angiotensin-I in the hypertensive rats fed only with distilled water, i.e. the NCG, was increased to a visually observable level compared to the normal rats, the PCG, and the treatment group. Therefore, it was observed that suppressing the protein expression of angiotensin-I had an antihypertensive effect on the treated group.

Fig. 2. Experiment to confirm the expression of angiotensin-1 protein stained brown with 3,3′-diaminobenzidine in rat kidneys. Data are expressed as mean±SD. ^a–d In the same column are significantly different (p<0.05). Tissue sections were selected for the measurement of staining areas using ImageJ (bar=50 μm). NG, normal group (WKY) with distilled water; NCG, negative control group (SHR) with distilled water; PCG, positive control group (SHR) with Enalapril; LTG, low treatment group (SHR); MTG, medium treatment group (SHR); HTG, high treatment group (SHR); IHC, immunohistochemistry; WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats.

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