International Journal of Food Microbiology 113 (2007) 358 – 361 www.elsevier.com/locate/ijfoodmicro
Short communication
Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects T.D.T. Nguyen, J.H. Kang, M.S. Lee ⁎ Department of Microbiology, Pukyong National University, Busan 608-737, Republic of Korea Received 27 June 2005; received in revised form 3 January 2006; accepted 12 August 2006
Abstract The bacterium Lactobacillus plantarum PH04 was isolated from infant feces and tested positive for bile/acid tolerance and bile salt hydrolase activity. It was evaluated as a potential probiotic with cholesterol-lowering effect. Bile salt hydrolase activity was nine times greater in stationary phase than in exponential phase cells and increased when the cells were exposed to conjugated bile salts. L. plantarum PH04 was resistant to seven of nine antibiotics tested and did not produce β-glucuronidase. L. plantarum PH04 was fed to hypercholesterolemic mice at numbers of 107 CFU per mouse per day for 14 days. Compared with a control group, the serum cholesterol and triglycerides were respectively 7 and 10% lower in the group fed L. plantarum PH04, and fecal lactic acid bacteria increased while no any significant differences (P b 0.05) in body weight, visceral weigh index or bacteria translocation between two groups were observed. The results indicated that L. plantarum PH04 might be effective as a probiotic with cholesterol-lowering activities. © 2006 Elsevier B.V. All rights reserved. Keywords: Probiotics; Lactobacillus plantarum PH04; Bile salt hydrolase; Cholesterol-lowering effect
1. Introduction Hypercholesterol is a risk factor for cardiovascular disease, the leading cause of death in many countries (Law et al., 1994). It is therefore important to develop new ways of reducing serum cholesterol. Recently, lactic acid bacteria (LAB) have attracted attention as potential cholesterol-lowering milk additives (Chandan, 1999; Roos and Martin, 2000). LAB normally reside in the mouth and intestinal tract where they enhance immune responses (Ozawa et al., 1983; Fernandes and Shahani, 1990), exert antimutagenic and anticarcinogenic activities (Gilliland, 1989), and protect against gastrointestinal diseases (Kasper, 1998). The reduction of serum cholesterol could be an important health benefit of LAB, as a 1% reduction in serum cholesterol is associated with an estimated reductions of 2 to 3% in the risk of coronary artery disease (Manson et al., 1992). The reduction of cholesterol by LAB has been demonstrated in human, mouse, and pig studies (Kawase et al., 2000; Haberer et al., 2003). ⁎ Corresponding author. Tel.: +82 516206365; fax: +82 516116358. E-mail address:
[email protected] (M.S. Lee). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.08.015
Cholesterol-lowering effects may be due in part to the deconjugation of bile salts by strains of bacteria that produce the enzyme bile salt hydrolase (BSH; Taranto et al., 1997; Brashears et al., 1998; Pereira et al., 2003). As deconjugated bile salts are more readily excreted in the feces than conjugated bile salts (Gilliland and Walker, 1990; De Smet et al., 1994; De Rodas et al., 1996), bacteria with BSH activity may effectively reduce serum cholesterol by enhancing the excretion of bile salts, with a consequent increase in the synthesis of bile salts from serum cholesterol; or by decreasing the solubility of cholesterol, and thus reducing its uptake from the gut. In the present study, we identified and characterized a strain of LAB with BSH activity, and evaluated its potential as a cholesterol-reducing probiotic in mice. 2. Materials and methods 2.1. Bacterial isolation Fecal samples from newborn babies were collected at Ilsin Christian Hospital (Busan, Korea) and screened for bile and
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acid tolerance as previously described (Gorbach and Goldin, 1989). Bacteria were isolated by spreading fecal material onto Lactobacillus selection agar (LBS) plates (Difco Laboratories, Detroit, MI, USA) supplemented with 0.15% (w/v) oxgall (Difco). The plates were incubated under microaerobic conditions with 10% CO2, at 37 °C for 48 h. Bacteria that grew on the LBS plates were screened for the ability to grow in De Mann– Rogosa–Sharpe (MRS) broth (Difco), pH 3.0. Strains were considered acid-tolerant if they grew to ≥ 107 CFU/ml within 24 h. BSH activity was tested using the plate-assay described by Dashkevicz and Feighner (1989). Briefly, bacteria were grown on MRS agar plates supplemented with 0.5% (w/v) of the sodium salt of taurodeoxycholic acid (TDCA; Sigma, St. Louis, MO, USA) and 0.37 g/l CaCl2. The strain that showed the largest precipitation zone was selected for further characterization. 2.2. Strain identification and characterization The Lactobacillus strain used was identified based on Gram staining, morphology, and catalase activity. Scanning electron microscopy was performed as previously described (Yamauchi and Snel, 2000). The pattern of carbohydrate fermentation was determined using the API 50CHL kit (Biomerieux Vitek, Marcy L'Etoile, France), and the isolate was tested for the ability to grow at temperatures ranging from 5 to 60 °C, NaCl concentrations up to 10%, and pH values from 2 to 10. The isolate was tested for bile salt tolerance by assessing its viability when grown in MRS broth supplemented with 0%, 0.2%, or 0.4% oxgall (Difco). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein lysates was used to compare the isolated strain with reference Lactobacillus strains as previously described (Pot et al., 1993). For definite identification, 16S rRNA sequencing was performed using total DNA from the isolate. BSH activity was determined by measuring the amount of amino acid liberated from conjugated bile salts. From cultures grown for 24 h at 37 °C, cells were harvested by centrifugation at 6000 ×g for 10 min, washed twice with 0.1 M phosphate buffer, pH 6.0, and resuspended in the same buffer to obtain a suspension with an optical absorption at 600 nm (A600) of 3.0. The reaction mixture consisted of 350 μl of 0.1 M phosphate buffer, pH 6.0, 50 μl of 200 mM sodium salt of taurocholic acid (Sigma), and 200 μl of washed cell suspension. The tube was incubated at 37 °C for 20 min, then a sample (200 μl) was taken and 10 μl of 6 N HCl was added to terminate the reaction. The sample was centrifuged at 8000 ×g, for 5 min and the supernatant was collected for subsequently assaying for free amino groups by the ninhydrin reaction (Tanaka et al., 2000). A unit of BSH activity (U/ml) was defined as 1 nM of taurine released from the substrate in 1 min by 1 ml of culture broth. The effect of the growth phase on BSH activity was determined by inoculating MRS broth with 1% of a 24 h culture, and incubating at 37 °C for 24 h. At intervals of 3 h, portions of broth were withdrawn for determination of pH, cell numbers, and BSH activity. The isolate was tested for the production of β-glucuronidase using the API ZYM kit (Biomereux). Antibiotic resistance was assessed by disk
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diffusion assay using antibiotics disks (Becton Dickinson, Cockeysville, MD, USA). The antibiotics tested were erythromycin (E15), penicillin (P10), cefoxitin (CTT30), gentamicin (GM10), nalidixic acid (NA30), chloramphenicol (C30), ampicillin (AM10), tetracycline (TE30), and kanamycin (K30). 2.3. Animals and cholesterol analysis Lactobacillus plantarum PH04 was grown in MRS broth for 18 h at 37 °C to reach cell numbers of 109 CFU/ml. The cells were pelleted by centrifugation at 6000 ×g for 5 min, washed twice with sterile saline, then were resuspended in 0.85% NaCl to obtain numbers of approximately 107 CFU/25 μl dose. The suspension was prepared daily for feeding to mice. Twelve five- to six-week-old, ICR, male mice were housed in metal cages and maintained under temperature- and humidity-controlled conditions. They were fed a commercial diet ad libitum for 6 days. For the next 14 days, the mice were fed a high-cholesterol diet containing 10% w/v skim milk and 10% w/v cream, as previously described (Taranto et al., 1998). During the subsequent 14 days, doses of 107 CFU of L. plantarum pH04 were orally administered to half the mice each day. The remaining mice received no bacteria. The activity, behavior, and general health of the mice were monitored daily. Food and water intake and body weight were measured weekly. Numbers of bacteria in fecal matter were determined as CFU/g wet weight of feces before and after administration of bacteria for 14 days. Following the 14 days of bacterial treatment, the mice were euthanized with ether. Blood was obtained from the arteria cervicalis and the viscera of the mice were tested for bacterial translocation. Serum samples were analyzed for total cholesterol and triglycerides using an Express Plus analyzer (Chiron Diagnostics, Emeryville, CA, USA). Translocation of bacteria to blood and tissues was assessed as previously described (Zhou et al., 2000). Briefly, the mesenteric lymph nodes (MLN), spleen, and liver were excised aseptically, and each organ was placed in 0.2 ml brain heart infusion (BHI) broth (Difco) and homogenized. Then 100 μl of
Fig. 1. Changes in cell numbers (▴), pH (•) and bile salt hydrolase activity (n) of the culture medium during growth of Lactobacillus plantarum PH04 in MRS broth at 37 °C.
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the organ suspensions and 15 μl of blood were plated on MRS agar plates, and the plates were incubated for 72 h at 37 °C. The presence of 10 or more microorganisms on a plate indicated that bacterial translocation had occurred. Experimental data are presented as the mean and standard errors of the mean. Paired t-tests were conducted using Microsoft Excel and SPSS 11.5 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Strain isolation and characterization The isolate was a rod-shaped, Gram-positive, catalasenegative bacterium that was able to grow at temperatures between 25 and 45 °C, pH values between 6 and 11, and NaCl concentrations up to 6%. The patterns of carbohydrate fermentation and SDS-PAGE analysis of the isolate were similar to those of the L. plantarum group. Partial sequencing of 16S rRNA for 447 nucleotides indicated 97% identity with L. plantarum LP3 (GenBank accession No. AY675256). Therefore the isolate was confirmed as a strain of L. plantarum, and was designated L. plantarum PH04. PH04 had bile salt hydrolase activity that was ninefold higher during the stationary phase than during the exponential phase of growth (Fig. 1). BSH activity of L. plantarum PH04 was higher when the cells were incubated with than without conjugated bile salts, and the activity was higher with glycineconjugated than with taurine-conjugated bile salts. Neither the deconjugated bile salts of cholic acid nor the amino acids taurine or glycine induced BSH activity. L. plantarum PH04 was resistant to tetracyline, chloraphenicol, penicillin, nalidixic acid, kanamycin, gentamicin, and cefotefan, but was sensitive to erythromycine and ampicillin. The organism did not induce β-glucuronidase. 3.2. In vivo cholesterol-lowering effect of L. plantarum PH04 Mice fed L. plantarum PH04 had 7% lower total cholesterol and 10% lower serum triglycerides compared to the control mice (Table 1). Mice treated with the organism had tenfold more lactic acid bacteria in their feces compared to controls (P b 0.05). No significant differences (P b 0.05) in body weight, food intake or visceral weight index or differences in behavior between the groups were noted. Bacterial translocation was similar for both groups, with single MLN, liver and spleen samples from each group being positive for translocation.
Table 1 Total serum cholesterol and triglyceride levels of groups of 6 mice treated or not treated with Lactobacillus plantarum pH04 Treatment group
Total cholesterol (mg/dl)
Triglycerides (mg/dl)
Mean (SE) a
Mean (SE)
Treated Not treated
163 (5.2) a 175 (5.8) b
141 (7.9) a 157 (15.1) b
a SE, means in the same column with different letters are significantly different (P b 0.05).
4. Discussion Characterization of L. plantarum PH04 showed that it was bile- and acid-tolerant. These characteristics are important in potential probiotics, as bile tolerance is required for bacterial growth and survival in the small intestine (Lee and Salminen, 1995) and acid tolerance is required for the bacteria to survive passage through the stomach (Henriksson et al., 1999), as well as to survive in food (Lee and Salminen, 1995). L. plantarum PH04 did not produce β-glucuronidase, a toxic enzyme which has been implicated in the formation of carcinogens (Borriello et al., 2003). In addition, L. plantarum PH04 was resistant to most of the tested antibiotics, suggesting that the organism would not be affected by therapies using these antibiotics and might help maintain the natural balance of intestinal microflora during antibiotic treatments. BSH activity was detected primarily during the stationary phase of growth of L. plantarum PH04, which is consistent with previous findings for another Lactobacillus species (Lundeen and Savage, 1990). In that study, the authors suggested that because lactobacilli are responsible for most BSH activity in the small intestine, the bacteria passing through the small intestine might be in a physiological state similar to the stationary phase. The BSH activity of L. plantarum PH04 was induced by conjugated bile salts but not by deconjugated bile salts, and was greater in the presence of glycine-conjugated than taurineconjugated bile salts. This may be an important property for the in vivo cholesterol-lowering activity of PH04, since glycineconjugated bile salts are the most abundant conjugated bile salts in the human small intestine (Du Toit et al., 1998). Oral administration of L. plantarum PH04 lowered total serum cholesterol levels in mice without any pathogenic side effects and without bacterial translocation. Assessment of pathogenicity is one important component of probiotic safety studies (Marteau et al., 1997; Zhou et al., 2000), the indicators for which include splenomegaly and hepatomegaly. None of these morphological changes was noted as a result of L. plantarum PH04 treatment, nor were these significant differences in the visceral weight indices of the lymph nodes, spleen, or liver. In addition, bacterial translocation, the process by which intestinal bacteria pass through the mucosal epithelium and invade other organs, was similar for the two groups. Translocation is another important safety consideration, as it may cause bacteremia, septicemia, and even multiple organ failure (Borriello et al., 2003). Serum triglycerides were also lowered as a result of the L. plantarum PH04 treatment, without affecting the structure and relative weight of the liver. This suggests that the hypolipemic effect of the bacteria may not be due to a redistribution of lipids from the plasma to the liver, but rather to decreased intestinal absorption of lipids or increased lipid catabolism (Taranto et al., 1998). The results of this study indicate that L. plantarum PH04 is a safe probiotic with the potential to reduce serum cholesterol and triglyceride levels. Further studies will be required to determine the mechanism underlying the cholesterol-lowering effect. It will also be necessary to test more animals, using varying doses
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