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Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content

Background Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. Methods In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20~30 years old) to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108~109 CFU/ml) were orally administered to SD rats (fed a high-cholesterol diet) every day for 2 weeks. Results B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p < 0.05), and slightly increased serum HDL. B. longum SPM1207 also increased fecal LAB levels and fecal water content, and reduced body weight and harmful intestinal enzyme activities. Conclusion Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.

Here, we evaluated the ability of Bifidobacteria spp. isolated from healthy Koreans (20~30 years old) to lower cholesterol, inhibit harmful enzyme activities, and control the fecal water content.

Bacterial strains
The origins of the strains used in this study are shown in Table 1. Isolation of Bifidobacteria was performed from fecal samples of healthy Koreans (20~30 years old) collected by BBL's anaerobic sample collection and transport system to maintain anaerobic conditions, and were used within 24 h. Fecal samples were serially diluted 10-fold from 10 -1 to 10 -8 , and 100 μl was spread onto selective BL agar containing 5% sheep blood. After 48 h of incubation in anaerobic conditions (90% N 2 , 5% H 2 , 5% CO 2 ) (Bactron Anaerobic Chamber, Sheldon Manufacturing Inc., USA) at 37°C, brown or reddish-brown colonies 2~3 mm in diameter were selected for further identification [29].
A fructose-6-phosphate phosphoketolase (F6PPK) test was performed [30] to ensure that the colonies selected were Bifidobacteria. To identify the isolated Bifidobacterium spp. at the species level, 16s rRNA sequencing was performed by Bioleaders (Daejeon, Korea).
In vitro cholesterol-lowering test MRS broth (pH7.0) (Difco, USA) containing 0.05% L-cysteine·HCl·H 2 O (w/v) was prepared and autoclaved at 121°C for 15 min. Soluble cholesterol (polyoxyethanyl-cholesterol sebacate, Sigma, USA) was added to the prepared MRS broth and filtered through a 0.45 μm Millipore filter. The inoculation volume was 15 μl of provisional probiotic bacterial culture (10 8~1 0 9 CFU/ ml) solution per 1 ml cholesterol-MRS broth, and that was anaerobically incubated at 37°C for 24 h. Uninoculated MRS broth was also incubated at 37°C for 24 h for the control.
Following incubation, bacterial cells were removed by centrifugation (3,000 rpm, 10 min), and the spent broth and uninoculated control broth were then assayed for their cholesterol content. The remaining volume of cholesterol in the cholesterol-MRS broth was determined by the method reported by Rudel and Morris with a small modification [31]. To measure the amount of cholesterol, the dye layer is observed at 560 nm.

Experimental animals and diets
A total of 24 Sprague-Dawley (SD) male rats (5-weekold) were purchased from Central Lab Animal Inc. (Korea), and were housed in a temperature-controlled animal room (22 ± 2°C) with a 12 h light/dark cycle and humidity 55 ± 5%. Food and water were freely supplied. The animals were randomly selected and assigned to three groups (8 rats per group) according to the type of diet. Group 1 was fed a normal diet. Group 2 was fed a high-cholesterol diet and saline (as control). Group 3 was fed a high-cholesterol diet and B. longum SPM1207 (the best strain at lowering cholesterol in vitro). The composition of high-cholesterol feed is shown in Table 2. All the rats were acclimatized to the respective diets for a week before the experiment started. Rats in groups 2 or 3 received daily administrations of 0.2 ml of saline or B. longum SPM1207 (10 8~1 0 9 CFU/ml), respectively, for 2 weeks. Body weight was monitored weekly and food consumption was monitored daily.

Analysis of blood serum
At the end of the experimental period of 3 weeks, blood samples from each animal were collected into tubes by cardiac puncture to determine the serum cholesterol level. Serum was separated from the blood by centrifugation at 3,500 rpm for 10 min. The total cholesterol, HDL-cholesterol, and LDL-cholesterol were analyzed by Samkwang Lab (Korea). Fecal sampling and bacteriological analysis Fecal samples were collected weekly to determine the number of LAB, harmful enzyme activity, and water content. Fecal samples were taken directly from the rectum by rectal stimulation and immediately transferred into sterile tubes and kept at 4°C. Total LAB counts was performed on MRS-agar and incubated at 37°C for 48 h under anaerobic conditions (90% N 2 , 5% H 2 , 5% CO 2 ). The numbers of colony forming units (CFU) are expressed as log 10 CFU per gram.
Harmful enzyme activity of rat intestinal microflora Enzyme activities related to colon cancer were tested in fecal samples of rats as previously described [32][33][34].
Tryptophanase activity assay Tryptophanase activity was assayed using 2.5 ml of a reaction mixture consisting of 0.2 ml of complete reagent solution (2.75 mg pyridoxal phosphate, 19.6 mg disodium EDTA dihydrate, and 10 mg bovine serum albumin in 100 ml of 0.05 M potassium phosphate buffer, pH 7.5), 0.2 ml of 20 mM tryptophan, and 0.1 ml of the enzyme solution (suspended fecal sample), incubated for 1 h at 37°C, and then stopped by adding 2 ml of color reagent solution (14.7 g p-dimethylaminobenzaldehyde in 52 ml H 2 SO 4 and 948 ml 95% ethanol). The stopped reaction mixture was centrifuged at 3,000 rpm for 10 min and enzyme activity was measured by monitoring absorbance at 550 nm.
Urease activity assay Urease activity was assayed using 0.5 ml of a reaction mixture consisting of 0.3 ml of urea substrate solution (4 mM urea in 20 mM sodium phosphate buffer, pH 7.0) and 0.1 ml of the enzyme solution (suspended fecal sample) incubated for 30 min at 37°C and then stopped by adding 0.1 ml of 1 N (NH 4 ) 2 SO 4 . Phenolnitroprusside reagent (1 ml) and alkaline hypochlorite reagent (NaClO, 1 ml) were added to the stopped reaction mixture and incubated for 20 min at 65°C. The reaction mixture was centrifuged at 3,000 rpm for 10 min. Enzyme activity was measured by monitoring absorbance at 630 nm.
b-glucosidase activity assay β-glucosidase activity was assayed using 2 ml of a reaction mixture consisting of 0.8 ml of 2 mM pnitrophenyl-β-D-glucopyranoside and 0.2 ml of the enzyme solution (suspended fecal sample), incubated for 30 min at 37°C, and then stopped by adding 1 ml of 0.5 N NaOH. The stopped reaction mixture was centrifuged at 3,000 rpm for 10 min. Enzyme activity was measured by monitoring absorbance at 405 nm.
b-glucuronidase activity assay β-glucuronidase activity was assayed using 2 ml of a reaction mixture consisting of 0.8 ml of 2 mM pnitrophenyl-β-D-glucuronide and 0.2 ml of the enzyme solution (suspended fecal sample), incubated for 30 min at 37°C, and then stopped by adding 1 ml of 0.5 N NaOH. The stopped reaction mixture was centrifuged at 3,000 rpm for 10 min. Enzyme activity was measured by monitoring absorbance at 405 nm.

Measurement of fecal water content
The water content of fecal samples was measured using a drying oven (105°C, 24 h). Fecal water content (%) is calculated by: where W wet and W dry are the weight of the fecal sample before and after drying in the oven.

Statistical Analysis
Results were expressed as mean ± standard deviation (SD). For statistical evaluation of data, one-way ANOVA was applied using SPSS 13.0 for Windows followed by post hoc comparisons using the Tukey's test. Differences were considered significant at p < 0.05.

Lowering of serum cholesterol in rats
We then tested the hypocholesterolemic effects of this LAB in rats fed a high-cholesterol diet. A high cholesterol diet increased serum cholesterol levels (Table 4). B. longum SPM1207 treatment reduced total cholesterol from 111.3 to 84.4 mg/dl and LDL-cholesterol levels from 33.3 to 23.5 mg/dl. In addition, B. longum SPM1207 slightly increased HDL-cholesterol levels, but did not significantly (Table 4).

Fecal water content, body weight, and bacteriological analysis
The high cholesterol diet caused dry feces, but B. longum SPM1207 treatment increased fecal water content ( Figure 2).
The high cholesterol diet also increased body weight after 2 weeks, but B. longum SPM1207 blocked this increase ( Figure 3). Fecal LAB counts were similar in all groups before the experimental diets, but a high-cholesterol diet lowered LAB counts. LAB administration increased fecal LAB counts from 5.3 log 10 CFU/g to 6.9 log 10 CFU/g, which was significantly higher than controls (Figure 4).
Inhibitory effect on harmful enzyme of rat intestinal microflora B. longum SPM1207 significantly inhibited tryptophanase and urease activities and slightly decreased βglucosidase and β-glucuronidase activities ( Figure 5).

Discussion
Cardiovascular disease is the most important cause of death in westernized countries, including Korea. In the United States, 10 million people suffer from ischemic coronary arterial diseases, and spend 115 billion dollars per year to treat it [19]. According to NHANES (the third national health and nation examination survey) data and NCEP (national cholesterol education program) guide, a half million people have died of ischemic cardiac disease. [19,35].
Hypercholesterolemia is strongly associated with coronary heart disease and arteriosclerosis [35][36][37][38], and decreasing serum cholesterol is an important treatment option. HDL-cholesterol can prevent arteriosclerosis by removing cholesterol from the blood stream, whereas LDL-cholesterol causes accumulation of cholesterol in blood vessels [35,39] Figure 1 Sequence analyses of the B. longum SPM1207-16s rRNA gene shows 99% identity with B. longum DJO10A (879 bp). containing lactic acid bacteria, can reduce cholesterol.
Here, B. longum SPM1207 isolated from healthy Korean feces had hypocholesterolemic effects in vitro and in experimental animals (from 345.0 mg/dl to 262.8 mg/ dl, and 111.3 mg/dl to 84.8 mg/dl, respectively). B. longum SPM1207 also slightly increased HDL-cholesterol levels, in agreement with other findings that decreased total cholesterol was accompanied by simultaneous increases of HDL-cholesterol [47,48].
Cholesterol reduction by lactic acid bacteria can be explained by five mechanisms [49][50][51][52][53][54]: (a) fermentation products of lactic acid bacteria inhibit cholesterol synthesis enzymes and thus reduce cholesterol production; (b) the bacteria facilitate the elimination of cholesterol in feces; (c) the bacteria inhibit the absorption of cholesterol back into the body by binding with  Effect of B. longum SPM1207 on fecal water content. All rats were acclimatized to the respective diets for a week. Then the rats in group 2 or 3 received daily 0.2 ml saline or B. longum SPM1207 (10 8~1 0 9 CFU/ml), respectively, for 2 weeks. Data are presented as means and standard deviation. *p < 0.05 significantly different compared with 1 week. Changes in body weight fed experimental diets for 3 weeks. All rats were acclimatized to the respective diets for a week. Then the rats in group 2 or 3 received daily 0.2 ml saline or B. longum SPM1207 (10 8~1 0 9 CFU/ml), respectively, for 2 weeks. Data are presented as means and standard deviation. *p < 0.05 significantly different compared with control (Cholesterol + Saline). Changes in total LAB counts in rats fed experimental diets for 3 weeks. All rats were acclimatized to the respective diets for a week. Then the rats in group 2 or 3 received daily 0.2 ml saline or B. longum SPM1207 (10 8~1 0 9 CFU/ml), respectively, for 2 weeks. Data are presented as means and standard deviation. *p < 0.05 significantly different compared with control (Cholesterol + Saline). cholesterol; (d) the bacteria interfere with the recycling of bile salt (a metabolic product of cholesterol) and facilitate its elimination, which raises the demand for bile salt made from cholesterol and thus results in body cholesterol consumption; and, (e) due to the assimilation of lactic acid.
Here, B. longum SPM1207 decreased tryptophanase, urease, β-glucosidase, and β-glucuronidase in rats. Fecal LAB counts in the B. longum SPM1207 feeding group was about 10 times greater than that in the control group, indicating bacterial survival through the gastrointestinal tract.

Conclusion
In conclusion, the incorporation of B. longum SPM1207 in the diet suppressed serum cholesterol levels on a cholesterol-enriched diet. This LAB also improved the balance of the intestinal flora, improved tryptophanase, urease, β-glucosidase, and β-glucuronidase, and increased fecal LAB levels and fecal water content. Therefore, B. longum SPM1207 may be a functional probiotic to treat hypercholesterolemia, help prevent colon cancer, and constipation. Studies in humans, however, could be resulted in contradictory outcomes. So, further clinical trials to confirm these effects must be conducted.