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Beneficial Microbes, 2015; 6(3): 287-293

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Effects of lactic acid bacteria on low-density lipoprotein susceptibility to oxidation and aortic fatty lesion formation in hyperlipidemic hamsters M. Ito1*, K. Oishi2, Y. Yoshida1, T. Okumura1, T. Sato1, E. Naito1, W. Yokoi1 and H. Sawada1 1Yakult

Central Institute, Yaho 1796, Kunitachi-shi, Tokyo, 186-8650 Japan; 2Yakult Honsha European Research Center for Microbiology ESV, Technologiepark 4, 9052 Gent-Zwijnaarde, Belgium; [email protected] Received: 10 April 2014 / Accepted: 22 September 2014 © 2014 Wageningen Academic Publishers

RESEARCH ARTICLE Abstract We investigated the effects of Streptococcus thermophilus YIT 2001, a strain of lactic acid bacteria, on the susceptibility of low-density lipoprotein (LDL) to oxidation and the formation of aortic fatty lesions in hyperlipidemic hamsters. S. thermophilus YIT 2001 had the highest in vitro antioxidative activity against LDL oxidation among the 79 strains of lactic acid bacteria and bifidobacteria tested, which was about twice that of S. thermophilus YIT 2084. The lag time of LDL oxidation in the YIT 2001 feeding group was significantly longer than in controls, but was unchanged in the YIT 2084 group. After the feeding of YIT 2001, lag times were prolonged and areas of aortic fatty lesions were dose-dependently attenuated, although there were no effects on plasma lipid levels. These results suggest that YIT 2001 has the potential to prevent the formation of aortic fatty lesions by inhibiting LDL oxidation. Keywords: LDL, antioxidative activity, atherosclerosis

1. Introduction Hyperlipidemia (hypercholesterolemia and hypertri­ glyceridemia) is a major risk factor for the development of atherosclerosis, and the oxidation of low-density lipoprotein (LDL) is recognised to play an important role in its initiation and progression (Ishigaki et al., 2008; Niki, 2011; Steinberg, 1997). Oxidised, but not native, LDL promotes vascular dysfunction by increasing the chemotactic properties of monocytes and transforming macrophages into foam cells through the scavenger receptor-mediated intake of oxidised LDL. Oxidised LDL also shows cytotoxic potential, which is probably responsible for endothelial cell damage and macrophage degeneration in the atherosclerotic plaque. Based on the oxidation hypothesis of atherosclerosis, dietary antioxidants have attracted considerable attention as preventive agents, and several structurally unrelated antioxidants inhibit the formation of atherosclerosis in animal models (Fruebis et al., 1999; Miyazaki et al., 2008; Ling et al., 2002; Vinson et al., 2001; Xu et al., 1998). A widely used ex vivo approach to estimate the oxidative susceptibility of LDL is to determine the duration of the lag

phase, the time prior to the rapid formation of conjugated dienes (propagation phase). The lag time is reported to be closely associated with the severity of coronary and carotid artery disease status (Aoki et al., 2012; Hendrickson et al., 2005; Regnström et al., 1992). In a hamster model of the effects of dietary manipulations on plasma lipoprotein metabolism (Spady and Dietschy, 1985; Srivastava and He, 2010), increases in dietary cholesterol and saturated fat led to elevated lipid and lipoprotein cholesterol plasma levels. Moreover, hyperlipidemic hamsters were shown to develop macrophage/foam cell-rich fatty streaks, the earliest recognisable lesion of atherosclerosis, in the aortic arch (Asami et al., 1999; Auger et al., 2002; Nistor et al., 1987; Srivastava and He, 2010). Lactic acid bacteria are commonly used as a starter of dairy products such as yogurt and have recently attracted attention because of their beneficial effects on human health. Kaizu et al. (1993) reported that the oral administration of lactic acid bacteria cellular extracts inhibits the hemolysis of erythrocytes in vitamin E-deficient rats. Lin and Yen (1999) found that Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus inhibit lipid peroxidation in

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0040287

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M. Ito et al.

linoleic acid emulsions, while Terahara et al. (2000) showed that the level of thiobarbituric acid-reactive substances is lower in the LDL of rats fed oxidised oil in conjunction with L. delbrueckii ssp. bulgaricus cultures than in control animals. We previously found that S. thermophilus YIT 2001 had the highest antioxidative activity against lipid peroxidation in liposomes out of 49 strains of lactic acid bacteria, and showed that the feeding of these bacterial cells attenuated oxidative injury in the colonic mucosa of iron-overloaded mice (Ito et al., 2003). However, the means by which S. thermophilus YIT 2001 protects against LDL oxidation is poorly understood. The present study aimed to investigate the effects of feeding S. thermophilus YIT 2001 on the susceptibility of LDL to oxidation compared with the lower antioxidative S. thermophilus YIT 2084 and on the formation of aortic fatty lesions in hyperlipidemic hamsters.

2. Materials and methods Bacteria and culture conditions Bifidobacterium (11 strains), Lactobacillus (27 strains), Lactococcus (2 strains), Leuconostoc (2 strains), and S. thermophilus (37 strains) were obtained from the Culture Collection Research Laboratory of the Yakult Central Institute for Microbiological Research (Tokyo, Japan). The bacteria were cultured in modified GAM broth (Nissui Seiyaku Co., Tokyo, Japan) containing 2% lactose for 18 h at 37 °C. Bacterial cells were then harvested and washed in saline by centrifugation at 3,000×g for 15 min at 4 °C. For animal experiments, the collected cells of S. thermophilus YIT 2001 or YIT 2084 were co-lyophilised with glucose and skimmed milk (dry weight ratio of cells:glucose:skimmed milk = 40:25:20).

Inhibition of low-density lipoprotein oxidation in vitro The cellular content of lactic acid bacteria was extracted in 80% ethanol. After drying under N2 gas, the extract was dissolved in phosphate-buffered saline (PBS). The LDL fraction (d=1.006-1.182 g/ml) was prepared from human serum (Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA) by density-gradient ultra-centrifugation (Himac CS120FX equipped with S120AT2 rotor; Hitachi, Tokyo, Japan) at 406,000×g for 2 h at 10 °C (Kano et al., 2005) and was dialysed with phosphate buffered saline (PBS) at 4 °C in anaerobic conditions. The lag time of LDL oxidation was measured by the method of Hirano et al. (1997). In brief, the oxidant 2,2’-azobis(4-methoxy)-2,4-dimethylvaleronitrile (V-70, 0.25 mM) was added to the reaction mixture containing the LDL fraction (final concentration of protein: 0.1 mg/ml) and bacterial extract (0.25 mg/ml) and incubated at 37 °C. The formation of conjugated dienes was then monitored at 234 nm absorbance by a Beckman Model 288

DU650 spectrophotometer (Beckman Coulter K.K., Tokyo, Japan) equipped with a 12-position automatic sample changer. The lag time was defined as the time between the addition of the oxidant and the time at which the conjugated diene increased. The extension rate of the lag time was calculated from the times obtained from the reaction mixtures containing bacterial extracts and PBS, respectively. The coefficient of variation for the extension rate was 10.9(%) for S. thermophilus YIT 2001 (n=5).

Preparation of bacterial cell fractions The washed cells of S. thermophilus YIT 2001 were suspended in PBS and broken by two passages through a French pressure cell (SLM Instruments, Urbana, IL, USA) operated at 1,500 psi. The cell wall fractions were collected by centrifugation at 8,000×g for 15 min. The supernatant was separated into cytoplasmic matrix and cell membrane fractions by ultracentrifugation at 116,000×g for 16 h. The cell wall and cell membrane fraction precipitates were suspended in PBS and their volumes were matched to that of the cytoplasmic matrix fraction. A portion of the cytoplasmic matrix fraction was filtered through an ultrafiltration membrane with a nominal molecular weight limit of 10 kDa (BIOMAX-10, Millipore Corp., Billerica, MA, USA). The lag time of LDL oxidation was measured as described above without ethanol extraction.

Animals and diets All animal experiments were conducted in conformity with the Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Notification No. 88 of 2006, Ministry of Environment, Japan) and were approved by the Ethical Committee for Animal Experiments of Yakult Central Institute for Microbiological Research (Tokyo, Japan). Seven-week-old male Golden Syrian hamsters were obtained from Clea Japan, Inc. (Tokyo, Japan). The hamsters had free access to food and water, and were accommodated in a room with controlled temperature (25 °C), humidity (55%), and light (12 h light/dark cycles) during the entire experimental period. After acclimation for 1 week in which they were fed a commercial diet (MF, Oriental Yeast Co. Ltd., Tokyo, Japan), the hamsters were randomly divided (n=7-8) into three groups for each experiment (Tables 1 and 2). The composition of the basal diets (modified AIN-76A) were α-corn starch 49.5%, casein 20%, sucrose 10%, coconut oil 10%, cellulose fibre 5%, AIN-76 mineral mix (American Institute of Nutrition, 1977) 3.5%, AIN-76 vitamin mix 1%, cholesterol 0.5%, DL-methionine 0.3% and choline bitartrate 0.2%. The experimental diets were composed of the basal diet, glucose (0.25%), skimmed milk (0.2%) and bacterial cells (0, 0.05 or 0.4%). The viable bacterial count in the experimental diets containing 0.4% bacterial cells Beneficial Microbes 6(3)



After the dietary period in experiments 1 and 2, hamsters were fasted for 18 h and blood was obtained from the abdominal aorta under Nembutal anaesthetic (Abbott Laboratories, Abbott Park, IL, USA). Blood was put into a plastic tube containing ethylenediamine-tetraacetic acid dipotassium salt dihydrate (EDTA-2K) (1.2 mg/ml) to prepare the plasma by centrifugation. Plasma was stored at -80 °C until analysis. Animals were euthanised by bloodletting under Nembutal anaesthetic.

Biochemical analysis The LDL fraction (d=1.006-1.182 g/ml) was prepared from the plasma of each hamster by density-gradient ultra-centrifugation as described above (Miyazaki et al., 2008). According to the method of Hirano et al. (1997), the lag time of LDL oxidation was measured as described above without the addition of bacterial extract. The LDL fraction protein concentration was determined with a BCA protein assay kit (Pierce Co., Rockford, IL, USA). Plasma triglyceride and total cholesterol levels were assayed using the Determiner TC555 (Kyowa Medics, Tokyo, Japan) and Triglyceride E-test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan) kits, respectively.

compared on the basis of the extension rate of lag time in an LDL oxidation reaction (Figure 1). S. thermophilus YIT 2001 had the highest activity, while the activity of S. thermophilus YIT 2084 was approximately half that of S. thermophilus YIT 2001.

Subcellular localisation of active ingredients For the analysis of subcellular localisation of active ingredients, the cells of S. thermophilus YIT 2001 were disrupted by a French pressure cell and fractionated by centrifugation. The inhibitory activity of LDL oxidation was mainly localised in the cytoplasmic matrix fraction (Figure 2). The cytoplasmic matrix fraction filtrate that had passed through an ultrafiltration membrane had an activity level nearly equal to that before filtration.

Effect on plasma lipids and susceptibility to low-density lipoprotein oxidation in hyperlipidemic hamsters Plasma levels of total cholesterol and triglyceride were two-fold higher in hamsters fed a high-fat and highcholesterol experimental diet (total cholesterol 261±55 mg/dl, triglyceride 498±181 mg/dl), showing hyperlipidemia (hypercholesterolemia and hypertriglyceridemia), than those fed a normal diet (total cholesterol 125±8 mg/dl,

S. thermophilus YIT 2001 S. thermophilus YIT 2084 other strains

Aortic fatty lesion analysis Measurement of the aortic fatty lesion area was performed according to the method of Asami et al. (1999). In brief, each aortic arch was removed under anaesthesia, incised, rinsed with saline, and stained with oil red O. As an index of lesion formation, a percentage of the surface-stained area in the aortic arch was measured by image analysis using the WinROOF Professional ver. 3.53 (Mitani Corporation, Tokyo, Japan) image analysis software package.

Statistical analysis Statistical analyses were performed using the SAS System for Windows (release 6.12; SAS Institute, Inc., Cary, NC, USA). Data were subjected to one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. Each data value in the animal experiments is expressed as the mean ± standard deviation.

3. Results Inhibition of low-density lipoprotein oxidation in vitro The in vitro antioxidative activity of cellular extracts from 79 strains of lactic acid bacteria (S. thermophilus, Lactococcus, Leuconostoc and Lactobacillus) and bifidobacteria was Beneficial Microbes 6(3)

80

Lag time extension rate of LDL oxidation reaction (%)

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was 3×108 cfu/g. Hamsters were fed experimental diets for 4 and 10 weeks in experiments 1 and 2, respectively. Body weights were measured weekly.

Protective effects of lactic acid bacteria on LDL oxidation

60

40

20

0

ST

Lc

Ls

Lb

Bf

Figure 1. In vitro antioxidative activity of lactic acid bacteria and bifidobacteria on the basis of lag time extension rates in low-density lipoprotein oxidation reaction. ST: Streptococcus thermophilus, Lc: Lactococcus, Ls: Leuconostoc, Lb: Lactobacillus, Bf: Bifidobacterium. 289

M. Ito et al. Table 1. Effect of Streptococcus thermophilus YIT 2001 or YIY 2084 on total cholesterol and triglyceride in plasma, lag time of low-density lipoprotein (LDL) oxidation, and body weight of hamsters.a

Lag time extension rate of LDL oxidation reaction (%)

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150

100

Total cholesterol (mg/dl) Triglyceride (mg/dl) Lag time of LDL oxidation (min) Body weight at the start of test (g) Body weight at the end of test (g)

50

0

cell wall

membrane

matrix

-50

Figure 2. Subcellular localisation of active ingredients of Streptococcus thermophilus YIT 2001. The cells of S. thermophilus YIT 2001 were disrupted by a French pressure cell and fractionated by centrifugation. The cell wall fraction and cell membrane fraction were suspended in phosphate buffered saline and the volumes of these fractions were matched to that of the cytoplasmic matrix fraction. LDL = low-density lipoprotein.

triglyceride 230±44 mg/dl). In hyperlipidemic hamsters, the effects of feeding S. thermophilus YIT 2001 and YIT 2084 were compared but neither had any significant effect on the total cholesterol and triglyceride plasma levels or on body weight (Table 1). The LDL oxidation lag time was significantly longer in the S. thermophilus YIT 2001 group than in the control group, but S. thermophilus YIT 2084 had no effect on the LDL oxidation lag time in hyperlipidemic hamsters.

Effect on the formation of aortic fatty lesions in hyperlipidemic hamsters The effects of different doses of S. thermophilus YIT 2001 (0.05 and 0.4% in the experimental diet) were next examined in hyperlipidemic hamsters, but no significant differences in total cholesterol and triglyceride plasma levels or body weight were observed between the control group, the 0.05% group, and the 0.4% group (Table 2). However, the feeding of S. thermophilus YIT 2001 dose-dependently prolonged the LDL oxidation lag time and dose-dependently attenuated the percent area of aortic fatty lesions (Figure 3). Figure 4 shows the representative photomicrographs of lipid accumulation on the surface of aortic arch stained with oil red O. There were almost no oil red O-stained lipid droplets in the section of hamster fed the normal diets (AIN-76A).

290

Control

YIT 2001 group

YIT 2084 group

261±55 498±181 46.9±5.7 129±6 147±10

259±40 432±146 56.7±9.4b 129±6 152±10

255±26 448±100 49.1±5.8 128±5 151±8

a

Hamsters were fed either a control diet (Control), a diet containing 0.4% S. thermophilus YIT 2001 (YIT 2001), or a diet containing 0.4% S. thermophilus YIT 2084 (YIT 2084). All diets contained 10% coconut oil and 0.5% cholesterol. b Significant difference from the control group (P

Effects of lactic acid bacteria on low-density lipoprotein susceptibility to oxidation and aortic fatty lesion formation in hyperlipidemic hamsters.

We investigated the effects of Streptococcus thermophilus YIT 2001, a strain of lactic acid bacteria, on the susceptibility of low-density lipoprotein...
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